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Genotyping of Rotavirus in children under five years of age in the Southern region of Ecuador
Abstract
A total of 341 fecal samples obtained from children under five years of age with acute diarrhea disease, attending two nursing homes in Loja city, Ecuador were studied for rotavirus diagnosis. Out of them, 56 (16%) were rotavirus positive and 33 samples were genotyped for the determination of genotypes G and P. The most frequent genotypes were G4 (42%), P [6] (36%) and +9 mixed combination G4 P [6] (21%). This is the first molecular study on rotavirus carried out in Southern Ecuador and the obtained data confirms the variability of the rotavirus circulating strains in Ecuador.
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Summary. A study on the prevalence of rotavirus G and P genotypes was carried
out based on 253 stool specimens obtained from children living in the
Colombia northern coast region who were less than 3-years-old and who suffered
from acute diarrhea. A previous study had detected the presence of rotavirus A in
90 (36.5%) of the 246 samples tested by enzyme immunoassay (EIA), and these
strains were investigated in the present study. Of these, 50 strains yielded an RNA
electropherotype, most of which (80.0%) had long profiles and 20.0% of which had
short profiles. Genotyping of 84 positive samples indicated that 67.9% of the
strains could be typed. G1 (57.9%), was the most predominant VP7 genotype, followed
by G3 (21.1%), G9 (15.8%) and G2 (5.3%). Among the VP4 genotypes, P[4]
(49.1%) was the most prevalent, followed by P[6] 36.4% and P[8] (14.5%). Neither
G4 nor G8 nor P[9] types were detected. The most common G-P combinations
were G3 P[4] (8.8%) and G9 P[6] (7.0%), followed by G1 P[4] and G1 P[8] (5.3%
each). All G1 P[8] strains showed long RNA profiles, whereas G3 P[4] and G9 P[6]
displayed both long and short patterns. Mixed infections involved 21.0% of strains.
There was a marked diversity among strains collected, and novel strains, including
G9, as well as other atypical combinations of G and P genotypes, such as G9 P[6]
and G3 P[4], were found. [Int Microbiol 2004; 7(2):113–120]
We studied the transmission of rotavirus infection in households in peri-urban Ecuador in the vaccination era.
Stool samples were collected from household contacts of child rotavirus cases, diarrhea controls and healthy controls following presentation of the index child to health facilities. Rotavirus infection status of contacts was determined by RT-qPCR. We examined factors associated with transmissibility (index-case characteristics) and susceptibility (household-contact characteristics).
Amongst cases, diarrhea controls and healthy control household contacts, infection attack rates (iAR) were 55%, 8% and 2%, (n = 137, 130, 137) respectively. iARs were higher from index cases with vomiting, and amongst siblings. Disease ARs were higher when the index child was <18 months and had vomiting, with household contact <10 years and those sharing a room with the index case being more susceptible. We found no evidence of asymptomatic infections leading to disease transmission.
Transmission rates of rotavirus are high in households with an infected child, while background infections are rare. We have identified factors associated with transmission (vomiting/young age of index case) and susceptibility (young age/sharing a room/being a sibling of the index case). Vaccination may lead to indirect benefits by averting episodes or reducing symptoms in vaccinees.
In developing countries where diarrheal disease is a leading cause of morbidity and mortality in children under 5 years of age, enteric coinfection is common. There is little understanding, however, of the biologic interaction between coinfecting pathogens. The authors investigated the potential for synergistic interaction between coinfecting pathogens on diarrhea pathogenesis using an epidemiologic framework. They conducted community-based, case-control studies in 22 communities in northwestern Ecuador between 2003 and 2008. Risk ratios of diarrhea associated with single infections and coinfections were estimated. Interaction between coinfecting pathogens was assessed through departure from risk ratio additivity and multiplicativity after adjustment for age. On the additive scale, the authors found departure from the null value of 0 for rotavirus-Giardia coinfections (interaction contrast ratio = 8.0, 95% confidence interval: 3.1, 18.9) and for rotavirus-Escherichia coli coinfections (interaction contrast ratio = 9.9, 95% confidence interval: 2.6, 28.4). On the multiplicative scale, they found departure from the value of 1 for rotavirus-Giardia coinfections (multiplicative interaction = 3.6, 95% confidence interval: 1.3, 8.7). This research provides epidemiologic evidence for synergism between rotavirus and other enteric pathogens. During coinfection, the pathogenic potential of each organism appears to be enhanced. The potential for pathogenesis to be more severe in the presence of a rotavirus coinfection amplifies the need for rotavirus vaccination.
To obtain more information about rotavirus (ROTAV) genotypes in Burkina Faso, we characterized 100 ROTAVs isolated from fecal samples of children with acute gastroenteritis in the capital city of Ouagadougou, during December 2009-March 2010. Of note, 13% of the ROTAV-positive samples, including those with mixed infections, were positive for the unusual G6 genotype ROTAV strain. The genotypes identified were G9P[8], G6P[6], G1P[6], G3P[6], G1P[8], and G2P[4]. G9P[8] subgroup (SG)II strains dominated during the beginning of the ROTAV season, but later in the season, other G types associated with P[6] and SGI specificity emerged. This emergence was related to a shift in the overall age of infected children; ROTAV SGII infected younger children and induced more severe symptoms. The finding of a high incidence of G6P[6] strains highlights the need for long-term surveillance of ROTAV strains in Burkina Faso, especially when ROTAV vaccination is being considered in several African countries.
In this study 2,089 fecal samples from patients with gastroenteritis were analyzed from different hospitals in Panama, Costa Rica, and the Dominican Republic during the period comprised between December 2002 and July 2003. One hundred samples per country from the positives to the enzyme-linked immunosorbent assay (ELISA) kit were analyzed by reverse transcription-polymerase chain reaction (RT-PCR) to determine the G and P genotypes: in Panama, Costa Rica, and Dominican Republic the combinations G and P have a great diversity and unusual genotypes. These results highlight an unexpected diversity among rotavirus strains in these countries and emphasize the need for further serologic and genetic surveys on more rotavirus strains in Central America and the Caribbean. In this context, the next generation of rotavirus vaccines will need to provide adequate protection against diseases caused by unusual genotypes. These results represent the second report of rotavirus genotypes in Costa Rica and first-time reports of rotavirus genotypes in Panama and the Dominican Republic.
To assess the disease burden and characterize the epidemiology of rotavirus diarrhea in Latin America.
We conducted a literature review of studies of children < 5 years of age who were hospitalized or seen as outpatients for diarrhea and for whom rotavirus was sought as the etiologic agent of the diarrhea. This review included inpatient and outpatient studies published since 1998 that included at least 100 children and reported surveillance activities lasting at least 12 consecutive months.
A total of 18 inpatient and 10 outpatient studies met the criteria for inclusion in this review. Rotavirus was detected in a median of 31% of inpatients (range, 16%-52%) and 30.5% of outpatients (range, 4%-42%). The median detection rate was higher in studies that used an enzyme-linked immunosorbent assay (ELISA) (inpatients 38%, outpatients 33%) versus less sensitive methods of detection. The age distribution of rotavirus disease varied among countries, with 65%-85% of children hospitalized in the first year of life. Most countries had rotavirus admissions year round, and rotavirus generally exhibited a winter seasonal peak in both temperate and tropical climates.
The heavy burden of disease attributable to rotavirus in Latin America suggests that vaccines currently being tested could have considerable impact in preventing hospitalizations, clinic visits, and deaths. The findings of the young age distribution of patients highlight the importance of early immunization for the success of a vaccine program. The data suggest that future surveillance for rotavirus diarrhea in Latin America should use a standardized surveillance protocol with an ELISA for detection. Data from surveillance studies will be critical to monitor the impact of the future introduction of vaccines.
During the past decade, rotavirus genotype G9 has spread throughout the world, adding to and sometimes supplanting the common genotypes G1-G4. We report evidence of this spread in a population sample within rural Ecuador. A total of 1,656 stool samples were collected from both patients with diarrhea and from asymptomatic residents in 22 remote communities in northwestern Ecuador from August 2003 through February 2006. Rotavirus was detected in 23.4% of case-patients and 3.2% of controls. From these 136 rotavirus-positive samples, a subset of 47 were genotyped; 72% were of genotype G9, and 62% were genotype P[8]G9. As a comparison, 29 rotavirus-positive stool samples were collected from a hospital in Quito during March 2006 and genotyped; 86% were of genotype P[8]G9. Few countries have reported P[8]G9 rotavirus detection rates as high as those of the current study. This growing prevalence may require changes to current vaccination programs to include coverage for this genotype.
In Latin America and the Caribbean, rotavirus causes approximately 15,000 deaths, 75,000 hospitalizations, 2 million clinic visits and 10 million cases of rotavirus diarrhea annually. Two safe vaccines are available that are effective in preventing severe illness. To date, seven countries in Latin America (Brazil, Ecuador, El Salvador, Panama, Mexico, Nicaragua and Venezuela) have introduced the vaccine. For successful rotavirus vaccine introduction, the lessons learned re-emphasize the critical need for countries to have precise plans that will ensure technical, programmatic and financial sustainability of vaccine introduction. Of these lessons learned, programmatic feasibility and financial sustainability were particularly challenging for countries that were the first to introduce a rotavirus vaccine.
Polyacrylamide gel electrophoresis of rotavirus RNA has been used to provide diagnostically and epidemiologically useful information about avian and mammalian rotavirus infections. (1) Rotaviruses possessing four different electropherotypes were detected in a longitudinal survey of a 34,000 bird broiler flock. Two of these had not been described previously in chickens. When virus recognisates were grouped according to electropherotype, infection with different electropherotypes was seen to occur in waves, with each wave lasting about one week. Cross-immunofluorescence indicated that rotaviruses with different electropherotypes were antigenically distinct, with only one sharing an antigen with conventional mammalian rotaviruses. (2) Mich intra-species variation was observed when bovine and porcine rotavirus RNAs obtained from different farms were analysed on high resolution 12.5% polyacrylamide gels. Consistent differences were observed between bovine and porcine rotavirus RNA profiles. The detection of additional bands in some RNA profiles indicated that more than one population of rotavirus was present in some faeces specimens. The finding that only one electropherotype was observed in samples taken from heifer calves from a “closed” dairy farm over a 16 month period suggested that only one rotavirus was present and that its genome was electrophoretically stable. (3) One electrophoretically “atypical” recognisate was observed out of 70 samples from bovine or porcine clinical cases.
The Pan-American Health Organization established a rotavirus pre-vaccination disease burden and strain surveillance network
in Latin America and the Caribbean in 2004. During strain surveillance in Ecuador in 2005–2006, a rare rotavirus genotype,
G11P[6], was detected among common strains. Sequencing and phylogenetic analysis of this strain identified a novel lineage
of the G11 VP7 gene, most closely related to A253 (91.8% nt identity), a porcine rotavirus strain identified in Venezuela.
Most genes of this strain clustered with porcine, human-porcine or bovine-porcine reassortant strains; only VP6 and perhaps
NSP2 genes were more closely related to cognate genes of human rotaviruses. Thus, this strain was likely generated by gene
reassortment between porcine and human parental strains. Our study provides further evidence that animal rotaviruses play
an important role in genetic and antigenic diversity of rotaviruses pathogenic for humans.
WHO recommends routine use of rotavirus vaccines in all countries, particularly in those with high mortality attributable to diarrhoeal diseases. To establish the burden of life-threatening rotavirus disease before the introduction of a rotavirus vaccine, we aimed to update the estimated number of deaths worldwide in children younger than 5 years due to diarrhoea attributable to rotavirus infection.
We used PubMed to identify studies of at least 100 children younger than 5 years who had been admitted to hospital with diarrhoea. Additionally, we required the studies to have a data collection midpoint of the year 2000 or later, to be done in full-year increments, and to assesses diarrhoea attributable to rotavirus with EIAs or polyacrylamide gel electrophoresis. We also included data from countries that participated in the WHO-coordinated Global Rotavirus Surveillance Network (consisting of participating member states during 2009) and that met study criteria. For countries that have introduced a rotavirus vaccine into their national immunisation programmes, we excluded data subsequent to the introduction. We classified studies into one of five groups on the basis of region and the level of child mortality in the country in which the study was done. For each group, to obtain estimates of rotavirus-associated mortality, we multiplied the random-effect mean rotavirus detection rate by the 2008 diarrhoea-related mortality figures for countries in that group. We derived the worldwide mortality estimate by summing our regional estimates.
Worldwide in 2008, diarrhoea attributable to rotavirus infection resulted in 453,000 deaths (95% CI 420,000-494,000) in children younger than 5 years-37% of deaths attributable to diarrhoea and 5% of all deaths in children younger than 5 years. Five countries accounted for more than half of all deaths attributable to rotavirus infection: Democratic Republic of the Congo, Ethiopia, India, Nigeria, and Pakistan; India alone accounted for 22% of deaths (98,621 deaths).
Introduction of effective and available rotavirus vaccines could substantially affect worldwide deaths attributable to diarrhoea. Our new estimates can be used to advocate for rotavirus vaccine introduction and to monitor the effect of vaccination on mortality once introduced.
Previous studies suggest that the emerging G9P[8] genotype was the most prevalent rotavirus genotype in Ecuador during 2005. This present study provides a temporal analysis of the distribution of rotavirus genotypes in two locations within Ecuador by adding additional years (2006 - early 2008) to the originally reported 2005 data. Data were collected in a rural (northern coastal Ecuador) and urban (Quito) area. In the rural area, a community sample of cases (those presenting diarrhea) and controls (those not presenting diarrhea) were collected between August 2003 and March 2008 resulting in a total of 3,300 stool samples (876 cases and 2,424 controls). Of these samples, 260 were positive for rotavirus by an immunochromatographic test (196 cases and 64 controls). In Quito, 59 fecal samples were collected from children presenting diarrhea and diagnosed with rotavirus. An RT-PCR analysis of samples collected between 2005 and 2007 suggested that G9 was replaced by G1 and G2 in the rural and urban settings. During this period G9 decreased from 79% to 9% while G2 increased from 0% to 43% in the rural communities, and G9 decreased from 79% to 37% while G2 increased from 3% to 57% in the urban area of Quito. This rapid replacement of G9 by G1 and G2 reinforces the necessity of surveillance to inform vaccination programs.
Intermolecular interactions between polypeptide chains often play essential roles in such biological phenomena as replication, transcription, translation, transport, ligand binding, and assembly. We have initiated studies of the functions of the rotavirus SA114F gene 7 product by sequence analysis and expression in insect cells. This nonstructural protein, NS34, is a slightly acidic protein, and its secondary structure is predicted to be 78% alpha-helix, with several heptad repeats of hydrophobic amino acids being present in its carboxy half. NS34 was found in oligomers when analyzed in insect cells, in SA11-infected MA104 cells, and in cell-free translation reactions. Investigation of the multiple electrophoretically distinct forms of NS34 showed they were all composed of homooligomers. Deletion mutants constructed and tested for oligomerization showed that the carboxy terminus of the protein, containing the predicted heptad repeats, was responsible for oligomerization. A basic region present in NS34 of group A rotaviruses, found to be 40% conserved in NS34 of group C rotavirus, is a candidate for a functional domain of this protein. NS34, which was found to be associated with the cytoskeleton fraction of cells, also interacts with viral RNA. These results make it likely that NS34 plays a central role in the replication and assembly of genomic RNA structures.
The objective of the present study was to determine rotavirus etiology and prevalence of the different rotavirus serotypes in Ecuadorian children younger than 5 years of age with gastroenteritis. Children (729) less than 5 years of age with acute diarrhea from either public or private primary health care centers in 10 different provinces of Ecuador, between March 2006 and August 2006 were included in the study. Rotavirus infection was diagnosed using a commercial immunoenzymatic test. Rotavirus isolated from stool samples was genotyped. Rotavirus was detected in the feces of 269 of the 729 children (37%) with diarrhea. The most prevalent G genotypes were G9 (46.1%) and G2 (27.2%), while the predominant P genotypes were P[8] (57%) and P[4] (29.5%). Among the single infections, the predominant P/G combinations were: P[8]G9 (56.9%) and P[4]G2 (32.6%). The present countrywide survey is one of the major studies for one single season in Latin America and the first in its class in Ecuador. The value of expanding laboratory capability throughout Latin America in order to monitor rotavirus strains over time, with special attention directed at those strains obtained from children who experience vaccine failure, is critical. Only continuous monitoring of rotavirus disease burden and genotype surveillance will provide this information.
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