Journal of Clinical Virology

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1 Journal of Clinical Virology 55 (2012) Contents lists available at SciVerse ScienceDirect Journal of Clinical Virology j ourna l ho mepage: Distribution of rotavirus genotypes after vaccine introduction in the Triângulo Mineiro region of Brazil: 4-Year follow-up study A.C.B. Dulgheroff a, E.F. Figueiredo a, L.P. Moreira a, K.C. Moreira a, L.M.S. Moura a, V.S. Gouvêa b, A.L.S. Domingues a, a Instituto de Ciências Biológicas e Naturais, Microbiologia, Universidade Federal do Triângulo Mineiro, Uberaba, Minas Gerais, Brazil b Departamento de Virologia, Instituto de Microbiologia, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil a r t i c l e i n f o Article history: Received 21 December 2011 Received in revised form 9 June 2012 Accepted 11 June 2012 Keywords: Gastroenteritis Rotavirus Genotypes Vaccine a b s t r a c t Background: Rotaviruses are the major cause of diarrhea in children for which a monovalent G1P[8] vaccine has been provided free for all Brazilian infants since March Objectives: To investigate prevalence and genotypes of rotavirus strains causing diarrhea in children in Triângulo Mineiro, Minas Gerais, during , and to assess local vaccine impact. Study design: Fecal specimens were analyzed for rotavirus detection and characterization by PAGE, RT-PCR and PCR-genotyping assays. Results: Overall, rotavirus was diagnosed in 12.1% (76/630) cases, accounting for 35.8% of the hospitalizations and 6.5% of outpatient attendance due to diarrhea. A trend in rotavirus disease reduction occurred in both cities (71.8% and 83.4% in Uberaba; 95.3% in Uberlândia) up to 2009, but it reversed in 2010 with increased rotavirus cases in Uberlândia. Short pattern G2P[4] strains were detected in all but three (96%) cases of mixed/p[nt] infections with long electropherotypes. Conclusions: This 4-year follow-up study showed a reduction in rotavirus-related diarrhea and even skipped a rotavirus season, which is consistent with vaccine mediated protection. The rotavirus epidemic curve reflected the natural cyclic fluctuation of the single G2P[4] genotype, with sharp reduction of cases in 2008 leading to lack of a rotavirus 2009 season (both cases and hospitalizations) followed by its come back in Diarrhea cases related to either vaccine serotype/genotype (G1 or P[8]) were not detected. Thus, a new scenario emerged with a single epidemic genotype replacing the cocirculation of great diversity of genotypes, thus far, a hallmark of the epidemiology of rotavirus in Brazil Elsevier B.V. Open access under the Elsevier OA license. 1. Background Group A rotavirus, member of genus Rotavirus, family Reoviridae, is the major cause of acute infectious diarrhea in children and a serious public health problem in the world. 1 The use of an effective rotavirus vaccine to significantly reduce the incidence of severe disease has been advocated for decades. 2 The virus has a genome composed by 11 segments of double-stranded RNA with a high potential for reassortment during mixed infections, a relatively common event in enterically transmited diseases. 3 Abbreviations: PAGE, polyacrylamide gel electrophoresis; RT-PCR, reverse transcription-polymerase chain reaction; RNA, ribonucleic acid; VP, viral protein; NT, non-typed. Corresponding author at: Instituto de Ciências Biológicas e Naturais, Microbiologia, Universidade Federal do Triângulo Mineiro, Praç a Manoel Terra, 330 Bairro: N.S. Abadia, Uberaba, Minas Gerais, CEP: , Brazil. Tel.: ; fax: address: andredomingues@dcb.uftm.edu.br (A.L.S. Domingues). Rotavirus infects humans and several animal species producing diarrheal disease; presents a great diversity of serotypes which are based on a dual antigenic system composed by the outer capsid protein VP7 (G serotype) and the virus spike protein VP4 (P serotype); and both proteins independently elicit neutralizing antibodies. 1 Most epidemiologic surveys have used molecular assays (genotyping) to identify the rotavirus serotypes present in clinical specimens, and found that serotypes/genotypes G1P[8], G2P[4], G3P[8], G4P[8], and more recently, also G5P[8], G8P[6] and G9P[8] have been the ones responsible for major outbreaks or epidemics of diarrheal disease in humans. 1 On March 2006, an attenuated monovalent G1P[8] vaccine strain (Rotarix, GSK, Belgium) was introduced into the childhood immunization program of the Brazilian Public Health System (SUS). A survey on the frequency and distribution of rotavirus genotypes causing gastroenteritis in infants and children in the Triângulo Mineiro region of Minas Gerais has been underway since September Results from six months prior to and early after the launching of country-wide rotavirus immunization have been published. 4 Here Elsevier B.V. Open access under the Elsevier OA license.

2 68 A.C.B. Dulgheroff et al. / Journal of Clinical Virology 55 (2012) we report the results obtained during the 4-year follow up study ( ). 2. Objectives To investigate the prevalence and genotypes of rotavirus strains causing diarrhea in children in Triângulo Mineiro, Minas Gerais, during , and to investigate possible vaccine impact on the epidemiology of rotavirus gastroenteritis in the region. 3. Study design 3.1. Patients and specimens Fecal specimens from children up to ten years of age, clinically diagnosed with acute diarrhea were collected in two centers: Laboratório Jorge Furtado Medicina Diagnóstica (LJF) in Uberaba and IPAC Medicina Diagnóstica (IPAC) in Uberlândia. These laboratories collect and analyze specimens from private and public hospitals and pediatric clinics from the Triângulo Mineiro region. Samples from Uberaba represent the period and from Uberlândia the period. Uberaba ( 296,000 inhabitants) and Uberlândia ( 600,000 inhabitants) are the largest cities of the Triângulo Mineiro region in West Minas Gerais. They are approximately 100 km apart from each other, share high living standards and other social, economical and environmental conditions, but differ in other features. Although presently booming, older, historic and rural Uberaba is an important center of grain production and cattle breeding, in contrast to the urban, industrial and serviceoriented Uberlândia Rotavirus detection and characterization Fecal specimens were submitted to nucleic acid extraction, 5 PAGE 6,7 and silver staining. 8 Positive specimens were characterized by RT-PCR amplification, and PCR-typing assays Results A total of 630 specimens were received between April 2007 and December 2010: all were tested for rotavirus. They were collected continuously from April 2007 to December 2009 in Uberaba and from May 2008 to December 2010 in Uberlândia (Fig. 1A and B). Overall, 76 (12.1%) specimens were positive for rotavirus, all detected during the coldest and dry period of each year. Rotavirus contributed with 35.8% of diarrhea hospitalizations and 6.5% of Fig. 1. (A) Temporal distribution of diarrhea and rotavirus-associated cases in Uberaba, (B) Temporal distribution of diarrhea and rotavirus-associated cases in Uberlândia,

3 A.C.B. Dulgheroff et al. / Journal of Clinical Virology 55 (2012) Table 1 Distribution of acute gastroenteritis and rotavirus-related cases in hospitalized patients and outpatients in Uberaba during rotavirus season (May to December), and non-rotavirus season (January to April), Period No. positive samples/no. samples tested (%) Inpatient Outpatient Total 2007 May December 24/60 (40.0) 9/33 (27.3) 33/93 (35.5) January April a 0/2 (0.0) 0/3 (0.0) 0/5 (0.0) 2008 May December 1/5 (20.0) 4/45 (8.9) 5/50 (10.0) January April 0/1 (0.0) 0/27 (0.0) 0/28 (0.0) 2009 May October 0/0 (0.0) 1/17 (5.9) 1/17 (5.9) January April 0/6 (0.0) 0/15 (0.0) 0/21 (0.0) Total 25/74 (33.8) 14/140 (10.0) 39/214(18.2) Percentage of samples >48 Age groups (months) Fig. 2. Percentage of diarrheal disease according to patients age group in the Triângulo Mineiro region, MG. a There were no samples from January to March. outpatient attendance for diarrhea in both cities during the study period. Unexpectedly large numbers of diarrhea cases were seen during the summer months (January March), a non-rotavirus period, in which no rotavirus case was recorded. Thus, only comparable periods (May December) comprising the rotavirus seasons were considered for prevalence calculations. The distribution of samples and prevalence of rotavirus-associated diarrhea in hospitalized and non-hospitalized patients in the two collection centers are shown in Tables 1 and 2. There was a substantial decrease in the prevalence of rotavirus cases during the rotavirus seasons in both cities up to 2009: it dropped from 35.5% in 2007 to 10% in 2008 and 5.9% in 2009 in Uberaba; and from 19% in 2008 to 0.9% in 2009 in Uberlândia. However, this trend was reversed the following year in Uberlândia with an increased rotavirus prevalence of 20.5% in Overall, for the two sites combined, rotavirus contribution to hospital admissions was 42.2% (43/102) whereas rotavirus comprised only 9.2% (33/357) of the outpatient cases of gastroenteritis during the rotavirus seasons. Children aged 7 12 month-old composed the group most affected by acute diarrheal disease regardless of the cause (Fig. 2). However, the distribution of rotavirus-related cases varied somewhat over time showing a tendency to affect older children, especially in 2007 and 2008 (Fig. 3). All rotavirus-positive specimens presented characteristic group A electrophoretic patterns: 73 (96%) samples showed short pattern with at least three variants, and only three samples showed long patterns, each one showing a unique profile. The short pattern samples were all identified as genotype G2P[4] whereas the long profiles corresponded to mixed infections or incompletely typed strains and included the only two rotavirus cases detected in 2009: a G5G10P[NT] in Uberaba and a G3P[NT] in Uberlândia; Table 2 Distribution of acute gastroenteritis and rotavirus-related cases in hospitalized patients and outpatients from Uberlândia during rotavirus season (May to December), and non-rotavirus season (January to April), Period No. positive samples/no. samples tested (%) Inpatient Outpatient Total 2008 May December 8/14 (57.1) 12/91 (13.2) 20/105 (19.0) January April a 0/0 () 0/0 (0.0) 0/0 (0.0) 2009 May December 0/12 (0.0) 1/104 (1.0) 1/116 (0.9) January April 0/5 (0.0) 0/40 (0.0) 0/45 (0.0) 2010 May December 10/11 (90.9) 6/67 (9.0) 16/78 (20.5) January April 0/4 (0.0) 0/68 (0.0) 0/72 (0.0) Total 18/46 (39.1) 19/370 (5.1) 37/416 (8.9) a There were no samples from January to April. Fig. 3. Percentage of rotavirus cases according to patients age group in the Triângulo Mineiro region, MG. the third long profile, a G3P[8]P[4], was detected in 2008 in Uberlândia. Those three cases were outpatients aged 3 4 years old who were not eligible for vaccination. 5. Discussion Rotavirus infections occurred in the winter months as usually observed for Southeastern Brazil 4,13 17 and most of the world. 1 Overall, the detection rate of rotavirus in this study (12.1%) was similar to the period (15%) in the same region 4 and within the range of 8 23% described in a recent review of 49 Brazilian studies. 18 Nevertheless, the present study showed a striking decline in rotavirus-associated cases over the three seasons in Uberaba, with reductions of 71.8% and 83.4% in 2008 and 2009, respectively, and over the first two seasons in Uberlândia, with 95.3% reduction in 2009 when compared to Thus, the year of 2009 was quite unique in the Triângulo Mineiro region for the lack of a rotavirus season. In contrast with this large reduction observed in rotavirus cases, the number of non-rotavirus diarrhea cases did not decreased over time in Uberlândia (Fig. 1B), eliminating the possibility of bias due to specimen collection. In the following 2010 season, however, Uberlândia experienced a resurgence of rotavirus-associated cases, going back to the 2008 level of rotavirus diarrhea cases and hospitalizations. A substantial reduction in rotavirus activity in one season followed by resurgence in the next season has also been reported in the United States after vaccine introduction. 19 Despite reduction in rotavirus-related cases, a significant number of non-rotavirus diarrheas were observed throughout the study period with no apparent seasonality or diminished frequency over time, reflecting the need to search for other agents of infantile gastroenteritis.

4 70 A.C.B. Dulgheroff et al. / Journal of Clinical Virology 55 (2012) Previous data on rotavirus-associated disease in Uberaba spanning from about 6 months before and 10 months after vaccine introduction have shown long pattern G1P[8], G4P[8], G9P[8] and G?P[8] strains co-circulating in and the emergence of short pattern G2P[4] strains by mid In the present followup study, the fluctuation of G2P[4] genotype was well defined against a background devoid of long pattern P[8] strains, reaching its epidemic peak in 2007 and waning afterward, completing a typical 2 3 year prevalence cycle for this serotype. Unfortunately, samples from Uberaba, were no longer available after The 3- year survey conducted in Uberlândia began later, but the results of the two overlapping years ( ) were very similar to those of Uberaba: genotype G2P[4] also predominated in 2008 and was undetected in 2009; however, in 2010, this genotype made a comeback, increasing the prevalence of rotavirus primarily in hospitalized patients. This probably represents an unexpected continuation of the G2P[4] epidemic cycle in the region. A 2 3-year period of predominance followed by longer 6 10-year interval of absence, or only sporadic detection, of G2P[4] strains was well described in Australia 20 and constitutes a hallmark of the epidemiology of distinct rotavirus serotypes. 1,21 The absence of diarrhea cases exclusively related to genotype P[8] strains (G1 or non-g1) in the current study seems to indicate vaccine protection against homotypic (P[8]) rotavirus, regardless of the G type. In fact, clinical trials have demonstrated high (>87%) efficacy of Rotarix vaccine to protect against rotaviruses of the most prevalent G types associated with P[8] Nevertheless, this potentially vaccine driven effect observed just one year (2007) into the program, and sustained for at least four years in the Triângulo Mineiro region was quite remarkable. The changing pattern of circulating rotavirus types, with greatly reduced diversity of strains associated with disease since 2007 may have important implications for the evolution of rotavirus strains. However, distinct results in vaccine impact on the distribution of rotavirus genotypes were reported from Australia after the introduction of Rotarix in ,26 : G1P[8] predominated (largely due to an outbreak in the Northern Territory in 2010) followed by G2P[4]; the other common genotypes (G3P[8], G4P[8], G9P[8]) continued to circulate marginally; uncommon genotype combinations were detected more frequently than in pre-vaccine periods, thus suggesting a local increase in strain diversity. Interestingly, genotype G2P[4] was also the second most prevalent genotype detected in those Australian states that were using another rotavirus vaccine (Rotateq) in their program. Thus, emergence of G2P[4] in Australia 25 and other countries, as in Brazil, 4,15,30 33 most likely follows a global trend dictated by the oscillatory fluctuations of rotavirus genotypes seemingly unrelated to vaccination. While efficacy of Rotarix against G2P[4] was low (42%) in clinical trials, 22,24 a follow-up study in Europe 23 demonstrated good efficacy against gastroenteritis caused by G2P[4] strains; and a recent clinical trial conducted in Africa provided further evidence that Rotarix affords similar protection against gastroenteritis caused by G1 and non-g1 rotavirus strains. 34 Likewise, a case control study from Recife, Brazil 35 reported 77% vaccine efficacy against G2P[4] hospitalizations of infants between 6 and 12 months of age, although vaccination did not change the risk of hospitalization in older children. Lack of information on child s vaccination status was a limitation in the current study, nevertheless, based on the child s age at illness, 50.7% of children infected with G2P[4] strains (1/3 of those infected in 2007; 2/3 in 2008; and all in 2010) were eligible for vaccination. The mean vaccine coverage rate was 86% for the state of Minas Gerais in the period ( There was a higher prevalence (42%) of rotavirus-related diarrhea in hospitalized patients than in outpatients (9.2%), confirming that rotavirus is the major contributor to hospitalization of children due to diarrhea. 1 A relatively high prevalence of rotavirus cases was observed in children over 24 months of age at the epidemic peak in 2007 and in 2008, which may reflect the recent introduction of G2P[4] in the region after an interval of absense, 4 thus finding a large number of older children previously unexposed to this serotype. It is also possible that older children were less likely to be vaccinated than younger children. In 2010, children between 13 and 48 months were also the most affected during the resurgence of G2P[4] in Uberlândia. Long profile non-g1, non-p[8] strains were detected in just three odd cases of untypable P and/or mixed infections presenting typical animal G genotypes that have been associated with outbreaks of gastroenteritis in humans in several parts of the world (G3), in Asia (G10), or in Brazil (G5). 1,36,37 Of interest, a mixed infection G3P[8]P[4] was identified in 2008 and a G3P[NT] strain in 2009, both in Uberlândia; they may represent thresholds of still unsuccessful reassortants or recent animal transmission. This 4-year follow-up study showed a reduction in rotavirusrelated diarrhea and even skipped a rotavirus season (2009), which is consistent with vaccine mediated protection. A new scenario emerged with the disappearance of homotypic (G1 or P[8]) strains related to diarrhea cases, thus reducing the great diversity of strains of distinct genotypes co-circulating simultaneously which, thus far, constituted a hallmark of the epidemiology of rotavirus in Brazil. 3,15 The coincidental rise and spread of G2P[4] in post-vaccination period resulted in a simplified epidemiology, resembling that described in the twentieth century for United States and Europe 1 but seldom seen in pre-vaccination cities in Brazil. The consequences of vaccination on the epidemiology of rotavirus infections are still unpredictable, taking into account the very diverse nature of the virus and its great ability to reassort. 3 Continued genotype surveillance should provide better understanding of rotavirus evolution and assessment of the vaccine program. Funding This project was supported by CNPq, and A.C.B. Dulgheroff received a M.S. fellowship from CAPES/REUNI, Brasília, Brazil. Competing interests None. Ethical approval This project was approved by the Ethical Committee of the Universidade Federal do Triângulo Mineiro (UFTM). Acknowledgments We thank the laboratories Jorge Furtado Medicina Diagnóstica (Uberaba) and IPAC Medicina Diagnóstica (Uberlândia). References 1. Estes MK, Kapikian AZ. Rotaviruses. In: Knipe DM, Howley PM, editors. Fields virology, vol. 2. Philadelphia: Lippincott Williams & Williams; p Bishop RF. Development of candidate rotavirus vaccines. Vaccine 1993;11: Gouvea V, Brantly M. Is rotavirus a population of reassortants? Trends Microbiol 1995;3: Domingues ALS, Morais AT, Cruz RL, Moreira LP, Gouvêa VS. Rotavirus-associated infantile diarrhea in Uberaba, Minas Gerais, on the wake of the Brazilian vaccination program. J Clin Virol 2008;43: Boom R, Sol CJ, Salimans MM, Jansen CL, Wertheim-van Dillen PM, van der Noordaa J. Rapid and simple method for purification of nucleic acids. J Clin Microbiol 1990;28: Laemmli UK. 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