Hospital-Based Surveillance and Analysis of Genotype Variation in Nicaragua After the Introduction of the Pentavalent Rotavirus Vaccine

Khawaja, Shazia MPH; Cardellino, Anna MPH, CPH; Mast, T. Christopher PhD, MSc

Pediatric Infectious Disease Journal:
doi: 10.1097/INF.0000000000000074
Vaccine Reports

Background: A live, attenuated, pentavalent rotavirus vaccine (RV5) was introduced in Nicaragua in 2006 through a 3-year partnership between Merck & Co, Inc., and the Nicaraguan Ministry of Health. Nicaragua was the first developing nation to include rotavirus vaccine in its national childhood vaccine program. To monitor the possibility of changing circulating rotavirus strains after the introduction of RV5, we determined the genotypes responsible for rotavirus gastroenteritis-related hospitalization during the first 3 postvaccine years.

Methods: Stool samples were collected from children with acute gastroenteritis who presented to any of 6 participating hospitals within 7–14 days of symptom onset. Samples positive for rotavirus antigen were analyzed for P and G genotypes using a reverse transcription polymerase chain reaction method.

Results: Overall, the predominant strains were G2P[4] (41.5%), G1P[8] (40.6%), G4P[8] (5.1%) and G3P[8] (4.7%). Strain predominance varied by season. During the 2007 season, G4P[8] (53.2%) and G2P[4] (40.5%) predominated. In the 2008 season, G2P[4] (77.9%) and G1P[8] (12.6%) were predominant, while in the 2009 season, G1P[8] (79.3%) and G3P[8] (7.8%) were predominant.

Conclusion: No new or unexpected strains were predominant in the years immediately following the introduction of RV5 into Nicaragua. RV5 does not appear to have substantially altered the historical pattern of seasonal fluctuation in rotavirus genotypes.

Author Information

Merck Sharp & Dohme Corp., Whitehouse Station, NJ.

Accepted for publication July 30, 2013.

Shazia Khawaja, Anna Cardellino and T. Christopher Mast are employees of Merck Sharp and Dohme Corp., a subsidiary of Merck and Co., Inc. Whitehouse Station, NJ. This work was supported by Merck Sharp and Dohme Corp., a subsidiary of Merck and Co., Inc. Whitehouse Station, NJ.

The authors have no other funding or conflicts of interest to disclose.

Address for correspondence: Shazia Khawaja, MPH, Associate Principal Scientist, Epidemiology, Merck Research Laboratories, PO Box 1000, North Wales, PA 19486. E-mail:

Article Outline

Rotavirus gastroenteritis (RGE) is responsible for approximately 453,000 annual deaths worldwide in children <5 years of age, with RGE-related mortality occurring primarily in developing nations.1,2 Nicaragua is a low-income developing nation with an average estimated 41,122 outpatient visits, 4460 hospitalizations and 60 deaths due to rotavirus disease per year reported from 2001 to 2005 in children <5 years of age.3

The rotavirus season in Nicaragua is temporally similar to that of the United States and other Central American countries. The season begins in October, peaks between February and April and ends in June.4 Up to 3 rotavirus strains have been detected during a single season, but 1 typically predominates.4 The 2005 rotavirus season was characterized by a large, nationwide outbreak of RGE.5 More than 64,000 people were suspected to have been infected and at least 56 died; most of them were children.

Rotavirus has a dual classification system based on capsid proteins VP4 and VP7. Remarkable diversity exists: a recent review of data from 1996 to 2007 documented approximately 110,000 genotypes worldwide.6 However, 5 strains are associated with 80–90% of childhood rotavirus disease globally: G1P[8], G2P[4], G3P[8], G4P[8] and G9P[8].6,7 In 2006, a live, pentavalent, bovine-human reassortant vaccine (RV5; RotaTeq; Merck & Co., Inc., Whitehouse Station, NJ) was licensed in the United States to be administered to infants in a 3-dose schedule at 2, 4 and 6 months of age. RV5 is directed against rotavirus strains G1–G4 and P8. Several postlicensure studies of RV5 during the 2007–2008 rotavirus season demonstrated that the vaccine was highly effective and well-tolerated in the United States.8–10

Compelled by the severity of the 2005 rotavirus outbreak and other surveillance data,11 the Nicaraguan Ministry of Health partnered with Merck & Co., Inc., in 2006 with the goal of vaccinating every eligible child born in Nicaragua over a 3-year period (2006–2009) with RV5, and Nicaragua became the first developing nation to assess RV5 effectiveness.12 Approximately 1.3 million doses of RV5 were distributed to Nicaraguan children and administered as part of the routine national vaccine program. Two completed case-control studies using RV5 in Nicaragua demonstrated vaccine effectiveness for 3 doses of RV5 ranging from 58% to 76% against severe rotavirus in children <5 years of age.13,14 Nicaragua is supported by the Global Alliance for Vaccines and Immunization, an organization that assists eligible countries with their immunization programs,15 to address the long-term sustainability of the program. As a result, it was the first Global Alliance for Vaccines and Immunization-eligible nation to implement routine rotavirus vaccination into the national Expanded Program on Immunization. Of note, the 3-dose schedule of RV5 was administered at 2, 4 and 6 months of age in Nicaragua.

Reports in the literature have discussed the potential effects of rotavirus vaccination on genotype diversity—specifically, whether high coverage (>90%) of RV5 would allow atypical rotavirus strains to become more prevalent.7,16,17 This study sought to assess evidence of any potential change in rotavirus strains after RV5 introduction through a retrospective evaluation of genotype diversity from fecal samples collected in a surveillance study conducted in Nicaragua in the 3 years after RV5 was introduced into the routine national vaccine program.14

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Surveillance Study

Methods of the original surveillance study have been reported elsewhere.14 In brief, stool samples were collected as part of a prospective, observational, hospital-based, active surveillance study of RGE among children <5 years of age in Nicaragua from February 12, 2007 to October 9, 2009. The study included hospitals in the Western region of Nicaragua: León, Estelí, Chinandega, Granada and 2 sites in the capital city of Managua. Children included in the study were <5 years of age and presented to the emergency department or hospital with acute gastroenteritis within 72 hours of diarrhea onset. Acute gastroenteritis was defined as the first occurrence of ≥3 watery or looser-than-normal stools within a 24-hour period and/or forceful vomiting. Stool samples were collected within 7–14 days after the onset of symptoms and were shipped within 72 hours to a central storage and shipping facility in Managua.

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Specimen Assays

A validated enzyme immunoassay for detection of rotavirus antigen was performed at the Children’s Hospital Medical Center (Cincinnati, OH).18 Each rotavirus-positive sample was subsequently typed for VP4 and VP7 to detect P and G genotypes (Fig. 1), respectively, using a reverse transcription polymerase chain reaction at Pharmaceutical Product Development (Wayne, PA) using a method described elsewhere.19 Testing for most G types such as G1–G6, G8–G10, G12 and Bovine G6 were performed. No additional assays were done on nontypeable results.

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Statistical Analyses

Statistical analyses were primarily descriptive. The number and proportion of children enrolled with laboratory-confirmed RGE were determined. Rotavirus seasons were assigned to calendar years according to observed peak prevalence. Genotype outcomes were determined for samples with complete G- and P-type data and the results were stratified by year (Fig. 2).

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Ethical Approvals and Oversight

This study was reviewed by the ethical review committees of the Nicaraguan Ministry of Health and the National Autonomous University of León, Nicaragua. Written informed consent was obtained from parents or guardians of children before enrollment in the study.

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Patient Disposition and Rotavirus Antigen Results

The surveillance study enrolled 6174 children with acute gastroenteritis, of whom 6064 (98.2%) provided stool samples for testing. A total of 1082 (17.8%) had rotavirus-positive stool samples (Fig. 1). Over the 3 years of surveillance, 90 samples of RGE were confirmed in the 2007 season (February 12, 2007 through June 30, 2007), and 547 and 445 samples were confirmed in the 2008 and 2009 seasons (July 1, 2007 through June 30, 2008 and July 1, 2008 through October 9, 2009), respectively. Overall coverage for this study among all surveillance subjects eligible for vaccination was 80%.

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Genotype Results

A total of 1082 RGE-positive samples were characterized for G and P types, and 977 had either VP7 or VP4 data available for analysis (Fig. 1). Eight-hundred and seventeen samples were fully typed with results for both G- and P-type tests. One-hundred and five samples yielded either negative or nontypeable results for both VP7 and VP4, while 160 were partially typed (101 yielded VP4 but not VP7 results and 59 yielded VP7 but not VP4 results).

A total of 14 different G and P combinations were characterized. The most frequent strains observed were G2P[4], G1P[8], G4P[8] and G3P[8], which made up 92% of all characterized strains over the 31 months of follow-up. Strains G4P[6], G9P[8] and G1P[5] were less common with a combined frequency of 6%. The remaining 2% were made up of combinations such as but not limited to G2P[8], G1P[4] and G3P[4](Fig. 2).

Strain frequency varied by season. G4P[8] and G2P[4] were predominant during the 2007 season and together accounted for 94% of the rotavirus-positive samples represented. G2P[4] and G1P[8] were the most prevalent strains in the 2008 season, with frequencies of 78% and 13%, respectively. G1P[8] and G3P[8] were prevalent in the 2009 season, with a frequencies of 79% and 8%, respectively.

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From 2007 through 2009, the predominant rotavirus strains in Nicaragua following the introduction of RV5 into the routine national vaccine program were G2P[4], G1P[8], G4P[8] and G3P[8]. Small percentages of other strains were detected, and substantial variation in genotype frequency was observed by season.

These findings are similar to other surveillance studies in Nicaragua that found several genotypes circulating annually, but typically 1 strain predominating. Espinoza et al4 conducted a prevaccine study of rotavirus genotypes from 2001 through 2003 and found that the predominant strain in 2001 was G2P[4]. In 2002, G1P[8] was found to be the predominant strain. Interestingly, in 2003, G3P[8] emerged as the predominant strain after not having been detected in the 2001 season and being detected infrequently in 2002. Published data are limited for 2004 through 2006. However, G4P[8] was predominant among a small number (n = 27) of rotavirus-positive fecal samples collected during the 2005 rotavirus outbreak in Nicaragua (the last prevaccine season).20 In another small (n = 20) sample of specimens collected in Nicaragua during May and June of 2006, 60% were G4P[8] and 35% were G9P[8].21 Broader regional sampling indicated that the predominant strain depended upon the locale; in 2005, G1P[8] was predominant in Costa Rica and the Dominican Republic. In 2006, G9P[8] was predominant in Costa Rica while G2P[4] was predominant in Honduras. Overall, the available data demonstrate that temporal and regional fluctuations in rotavirus genotype occurred before the introduction of RV5 vaccine into Nicaragua. Moreover, the pentavalent rotavirus vaccine does not appear to have substantially altered the historical pattern of seasonal rotavirus genotypes in Nicaragua, for no new or unexpected strains were predominant in the years immediately following its introduction.

Pre- and postvaccine rotavirus genotype surveillance studies have also been conducted in the United States and Australia. In 2007, Australia introduced both RV5 and Rotarix (RV1; Glaxo SmithKline Biologicals, Rixensart, Belgium), a single, live, attenuated, human G1P[8] rotavirus strain,22 into the Australian National Immunization Program. For the first 2 years, 2008 and 2009, each vaccine was introduced into separate regions throughout Australia, providing the opportunity to determine which rotavirus genotypes were associated with hospitalization in a specific vaccine region.23 The vaccine coverage for both years was 80%. The 5 most common genotypes—G1P[8], G2P[4], G3P[9], G4P[8] and G9P[8]—accounted for 99% of all genotypes. However, some variation in genotypes was found between vaccine regions. The RV1 region had a comparatively increased frequency of the G2P[4] strain, whereas the RV5 regions had an increased frequency of G1P[8] and G3P[8]. Similar to the Nicaraguan data, prevaccine studies showed annual changes in the distribution of G2P[4], G3P[8], G4P[8] and G9P[8] genotypes. However, in the third postvaccine year in Australia (2009–2010), genotype G1P[8] reemerged as the dominant genotype nationally, representing 49.3% of all strains.24 Genotype G2P[4] was the second predominant type, comprising 21.1% of all strains characterized. The study concluded that continued fluctuations in rotavirus genotypes across Australia were minimally influenced by vaccination.

In the United States, the Rotavirus Strain Surveillance System monitored strain distribution for RV5 over 3 rotavirus seasons: 1 during the prevaccination period (2005–2006) and 2 during the postvaccination period (2006–2007 and 2007–2008).25 Each season was found to have 4 circulating genotypes: G1P[8], G2P[4], G3P[8] and G9P[8]. The predominant strain during the 2005–2006 and 2006–2007 seasons was G1. Little difference in strain distribution was seen in the 2006–2007 and 2007–2008 periods. However, during the 2007–2008 season, G3 emerged as the predominant strain. The authors cautioned against the interpretation that vaccine-related selective pressure played a role in the emergence of G3 during that season, owing to the context of overall dramatic decline in RGE and to the observation that the RV5 vaccine appeared to be highly effective against G3.

Comparing pre- and postvaccine genotype distribution is one approach to assessing the question of postvaccine strain emergence. Another approach is to examine the natural temporal variation in rotavirus strain distribution in countries without vaccine. For example, in Asian nations without rotavirus vaccine, increased frequencies of G2P[4] and G3P[8] have recently been reported.26,27 Changes in frequency of these rotavirus strains have been transient, lasting 1 or 2 years, and were followed by an increase in the prevalence of G1P[8], which is covered by both RV1 and RV5 vaccines. These fluctuations appear to be reflective of global trends: a review of worldwide data demonstrated considerable temporal and regional variability for the 7 most medically important G types from 1996 to 2007.6

Vaccine effectiveness studies across a variety of settings have demonstrated substantially reduced rotavirus hospitalizations and large declines in overall disease burden, despite considerable postvaccine variability in strain distribution.7,28–30 To date, vaccination does not appear to be associated with the emergence of predominant strains not covered by the available vaccines. Taking the available pre- and postvaccine data together, it appears unlikely that annual shifts in rotavirus strain distribution can be attributed to rotavirus vaccination.

This study may be limited by a lack of data from the time period immediately before introduction of the vaccine. However, the published prevaccine data from 2001 to 2006 provided a benchmark for the current work. In addition, the 3-year postvaccine follow-up period may not allow the evaluation of longer-term trends.

Full genotypes were obtained for approximately 76% of the samples in our study. Approximately 9% of samples yielded no genotype results, while approximately 15% were partially typed. Failure to type is typically caused by technical issues or low viral loads, and no further testing was done on partially typed samples.

This study of postvaccine rotavirus genotype distribution demonstrated that no unusual shifts in genotypes have occurred in Nicaragua in the 3 years since the introduction of RV5 into the national immunization program.

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The authors are grateful to the infants, parents and clinical physicians in Nicaragua for participating in the study. The authors thank medical writers Karen Collins and Eric Bertelsen, PhD, of Arbor Communications, Inc., and Tracey Fine, MS, ELS, of Fine Biomedical Publications, Inc., for assistance in drafting the manuscript, and Xingshu Zhu and April Grant for the statistical analysis and input from Merck. All authors were involved in the study design or collection, analysis and interpretation of data; in writing of the report; and in the decision to submit the article for publication.

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rotavirus; genotype; surveillance; vaccination; Nicaragua

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