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1.  Diversity of Group A Human Rotavirus Types Circulating over a 4-Year Period in Madrid, Spain 
Journal of Clinical Microbiology  2004;42(4):1609-1613.
The incidence and distribution of human rotavirus G types among children under 5 years old with acute gastroenteritis were determined over a 4-year period (1998 to 2002) by using monoclonal antibodies and reverse transcription-PCR methods. Rotavirus was detected in 1,155 (31%) of 3,760 specimens tested. Rotavirus was studied in every month of the 48-month survey period. Rotavirus activity occurred mainly (51%) in the typically cooler months in Spain (November to February). The age distribution of rotavirus-positive cases showed that 90% of patients (1,038 of 1,155) were under 2 years old. Rotavirus types were determined for 576 of 1,155 patients (50%). G1 was the main genotype detected (53%), and the second most common was G4 (24%). The G2, G9, and G3 rotavirus types were detected in 14, 6, and 2% of the cases, respectively. Dual infections were detected in only 0.6%. The seasonal distribution of genotypes showed a significant genotypic shift: whereas G4 strains predominated (57%) during the 1998 to 2000 seasons, the G1 gradually increased to account for 75% in the 2000 to 2002 seasons. In addition, the present study reports the first detection of the G9 genotype in human fecal samples in Spain. Therefore, additional types may be required for vaccine development strategies that currently target only types G1 to G4.
PMCID: PMC387563  PMID: 15071013
2.  Incidence of Human Astrovirus in Central Australia (1995 to 1998) and Comparison of Deduced Serotypes Detected from 1981 to 1998 
Journal of Clinical Microbiology  2002;40(11):4114-4120.
The incidence of astrovirus infection was determined among infants and young children admitted to Alice Springs Hospital (central Australia) with gastroenteritis from 1995 to 1998. Astrovirus was detected by reverse transcription-PCR in 33 of 495 stool samples, and this represented 4.3% of a total of 774 stool samples tested for astrovirus, rotavirus, and Norwalk-like viruses. Astrovirus incidence was substantially lower than that of rotavirus but higher than that of Norwalk-like viruses both overall and in each of the 4 years individually. Over the period from 1981 to 1998, including the period from 1981 to 1994 during which astrovirus was identified only by electron microscopy, astrovirus serotypes (deduced from genotypes) 1, 2, 3, and 4 were identified. Deduced serotypes 1, 3, and 4 all appeared regularly over this 18-year period. Also over this period, nucleotide variation (in some cases substantial) in a section of the capsid protein precursor region of the virus genome was evident among strains of all four of the deduced central Australian serotypes. Consequent amino acid changes were, however, only evident among deduced serotype 3 strains. Geographic variation at both the genome and the resultant amino acid levels was evident among strains of all four of the deduced central Australian serotypes and their respective prototypes isolated in the United Kingdom.
PMCID: PMC139637  PMID: 12409383
3.  Molecular Characterization of Rotavirus in Ireland: Detection of Novel Strains Circulating in the Population 
Journal of Clinical Microbiology  2000;38(9):3370-3374.
A collection of three hundred thirty rotavirus-positive stool samples from children with diarrhea in the southern and eastern regions of Ireland between 1997 and 1999 were submitted to the Molecular Diagnostics Unit of the Cork Institute of Technology, Cork, Ireland, for investigation. These strains were characterized by several methods, including polyacrylamide gel electropherotyping and G and P genotyping. A subset of the G types was confirmed by nucleic acid sequencing. The most prevalent types found in this collection included G1P[8] (n = 106; 32.1%), G2P[4] (n = 94; 28.5%), and G4P[8] (n = 37; 11.2%). Novel strains were also detected, including G1P[4] (n = 19; 5.8%), and G4P[4] (n = 2; 0.6%). Interestingly, mixed infections accounted for 18.8% (n = 62) of the total collection, with only 3% (n = 10) which were not G and/or P typeable. Significantly, six G8 and five G9 strains were identified as part of mixed infections. These strains have not previously been identified in Irish children, suggesting a greater diversity in rotavirus strains currently circulating in Ireland.
PMCID: PMC87388  PMID: 10970385
4.  Characterization of novel VP7, VP4, and VP6 genotypes of a previously untypeable group A rotavirus 
Virology  2009;385(1):58-67.
Rotavirus is the most common cause of acute gastroenteritis among infants and young children throughout the world, but rotavirus cases in developing countries account for nearly all of the ∼600,000 annual deaths. We studied the epidemiology of rotavirus in 22 rural communities in northern coastal Ecuador over a five-year period. From 250 rotavirus positive stool specimens, the percentage that could not be RT-PCR genotyped for VP4 and VP7 was 77% and 63%, respectively. The possibility of sample degradation was considered but discounted after an experimental examination of rotavirus stability and EM visualization of rotavirus-like particles in several untypeable samples. Finally, alternate primers were used to amplify Ecu534, a sample that was untypeable using most published VP4 and VP7 primers. Characterization of the VP7, VP4, and VP6 full gene segments revealed novel genotypes and nucleotide mismatches with most published primer sequences. When considered with other findings, our results suggest that primer mismatch may be a widespread cause of genotyping failure, and might be particularly problematic in countries with greater rotavirus diversity. The novel sequences described in this study have been given GenBank accession numbers EU805775 (VP7), EU805773 (VP4), EU805774 (VP6) and the RCWG has assigned them novel genotypes G20P[28]I13, respectively.
PMCID: PMC2729439  PMID: 19131083
rotavirus; novel genotype; genotyping failure; primer design; RT-PCR; Ecuador
5.  Genotyping Rotavirus RNA from Archived Rotavirus-Positive Rapid Test Strips 
Emerging Infectious Diseases  2011;17(1):44-48.
Genotyping circulating rotaviruses before and after introduction of rotavirus vaccine is useful for evaluating vaccine-associated changes in genotype distribution. We determined frequency of rotavirus genotypes among 61 rotavirus-positive children hospitalized in Israel during the 2005–06 rotavirus season. Accurate molecular epidemiologic data were recovered from affinity-concentrated rotavirus immobilized in rotavirus-positive bands from air-dried, diagnostic rotavirus rapid test strips (dipstick) stored at room temperature from 1 week to 5 years. G genotypes were identical for 21 paired dipsticks and suspensions, whereas dipsticks or suspensions detected an additional G genotype in 2 samples. RNA sequences from 7 pairs were identical. Phylogenetic analysis suggested previously unreported G2 sublineages and G9 lineages. The ease with which dipsticks can be stored at local facilities and transported to central reference laboratories can reverse increasing difficulties in obtaining geographically representative stool samples and expand surveillance to regions lacking adequate laboratory facilities.
PMCID: PMC3204647  PMID: 21192853
Rotavirus; genotyping; affinity purification; viruses; Israel; research
6.  Burden and Typing of Rotavirus Group A in Children with Acute Gastroenteritis in Shiraz, Southern Iran 
Human Rotavirus is a significant cause of severe gastroenteritis in infants and young children worldwide. In recent years, Rotavirus genotyping by RT-PCR has provided valuable information about the diversity of Rotaviruses circulating worldwide. The purpose of the present study is to monitor the prevalence of the different G types of Rotaviruses circulating in Shiraz, Southern Iran and detect any uncommon or novel types.
During the period from December 2007 to November 2008, a total of 138 stool samples were collected from children less than 5 years old who were hospitalized for acute gastroenteritis. Rotavirus-associated diarrhea was investigated in fecal specimens with enzyme immunoassays (EIA). Rotavirus-positive specimens were typed by the Nested RT-PCR and by using different types of specific primers.
Out of the 138 collected samples, 34.78% (48 cases) tested positive for Rotavirus. The frequency of G1, G2 and G4 types was 6.25%, 2.08% and 27.08%, respectively. Mixed and non-typeable infections were detected in 33.34% and 31.25% of hospitalized children with acute diarrhea, respectively. This is the first time mixed Rotavirus infections with G1/G3 have been reported in Iran.
The high frequency of Rotavirus detection indicates the severity and the burden of Rotavirus disease may be able to reduce through the implementation of an effective vaccine and continual surveillance for the detection of Rotavirus genotypes circulating in other regions of Iran.
Regarding to the noticeable frequency of non-typeable and mixed infections, it is suggested to use the other specific primers and further studies to detection of other novel and unusual types.
PMCID: PMC3482325  PMID: 23115715
Human Rotavirus; Diarrhea; Hospitalized children; G types
7.  Application of Restriction Fragment Length Polymorphism Analysis of VP7-Encoding Genes: Fine Comparison of Irish and Global Rotavirus Isolates 
Journal of Clinical Microbiology  2002;40(2):524-531.
A restriction fragment length polymorphism (RFLP) detection assay was developed to examine the genetic relationship(s) among VP7-encoding genes from 100 Irish rotavirus isolates and 30 randomly selected global rotavirus isolates (from the current databases). RFLP analysis of the VP7 gene segments was performed independently with three enzymes (RsaI, AluI, and EcoRV) in separate reactions by direct digestion of the DNA product amplified by reverse transcriptase (RT)-mediated PCR (RT-PCR) or by using computational methods. Thirty-six RFLP patterns were identified for all 130 strains, and of these, only nine patterns were associated with the Irish isolates. A correlation between the G type of the Irish isolates and certain single or combined enzyme profiles was apparent. These data suggested that the Irish wild-type rotavirus population was homogeneous and could be distinguished by RFLP analysis from global isolates of the same serotype(s). The deduced amino acid sequences of the VP7 RT-PCR products from six Irish isolates known to be of the G serotype revealed significant amino acid substitutions within major antigenic regions. In addition, these data identified the existence of at least two genetic lineages within serotype G1 strains which were distinguishable by RFLP analysis.
PMCID: PMC153395  PMID: 11825967
8.  Rotavirus genotypes associated with childhood severe acute diarrhoea in southern Ghana: a cross-sectional study 
Virology Journal  2013;10:287.
Rotavirus immunization has been effective in developed countries where genotype G1P[8] is the predominant rotavirus strain. Knowledge of circulating strains in a population before introduction of rotavirus immunization program will be useful in evaluating the effect of the intervention.
Rotavirus was identified by enzyme immuno-assay (EIA) on stool specimens of children (age 0 – 59 months) hospitalized with acute gastroenteritis from August 2007 to February 2011 in Accra, Ghana. Rotavirus positive specimens were further characterized by polyacrylamide gel electrophoresis (PAGE) and reverse-transcriptase polymerase chain reaction (RT-PCR).
Of the 2277 acute gastroenteritis hospitalizations 1099 (48.2%) were rotavirus-positive by EIA. Of the 1099 cases 977 (89%) were PAGE positive. All EIA positive specimens were further subjected to RT-PCR and 876 (79.7%) had sufficient material for characterization. Of these 876 cases, 741 (84.6%) were assigned G genotype, 709 (80.9%) P genotype, and 624 (71.2%) both G and P genotypes. We identified 8 G genotypes (G1, G2, G3, G4, G8, G9, G10, G12) and 3 P genotypes (P[4], P[6], P[8]). G1 (50.9%), G2 (18.8%), G3 (12.8%), P[8] (36.1%) and P[6] (30.7%) were the most prevalent. The most prevalent genotype combination was G1P[8] (28%). Mixed G (7.3%) and P (24.2%) genotypes were not uncommon. There was year-by-year and seasonal variations for most genotypes.
There is great diversity of rotavirus strains in children with severe gastroenteritis in southern Ghana. Even though cross-protection with vaccine-induced immunity occurs, continued strain surveillance is recommended after the introduction of rotavirus vaccine in the national immunization program.
PMCID: PMC3848793  PMID: 24034588
Rotavirus; Gastroenteritis; Diarrhoea; Genotypes; Ghana; Immunization
9.  Unexpectedly high burden of rotavirus gastroenteritis in very young infants 
BMC Pediatrics  2010;10:40.
The highest incidence of rotavirus gastroenteritis has generally been reported in children 6-24 months of age. Young infants are thought to be partially protected by maternal antibodies acquired transplacentally or via breast milk. The purpose of our study was to assess the age distribution of children with confirmed community-acquired rotavirus gastroenteritis presenting to an urban referral hospital.
Children presenting to The Children's Hospital of Philadelphia with acute gastroenteritis have been monitored for the presence of rotavirus antigen in the stool by ELISA (followed by genotyping if ELISA-positive) since the 1994-95 epidemic season.
Over the last 12 rotavirus seasons prior to the introduction of the pentavalent rotavirus vaccine in 2006, stool specimens from 1646 patients tested positive for community-acquired rotavirus infection. Gender or age was not recorded in 6 and 5 cases, respectively. Overall, 58% of the cases occurred in boys. G1 was the predominant VP7 serotype, accounting for 72% of cases. The median (IQR) age was 11 (5-21) months. A total of 790 (48%) cases occurred in children outside the commonly quoted peak age range, with 27% in infants <6 months of age and 21% in children >24 months of age. A total of 220 (13%) cases occurred during the first 3 months of life, and the highest number of episodes per month of age [97 (6%)] was observed during the second month of life.
The incidence of community-acquired rotavirus gastroenteritis monitored over 12 seasons in the prevaccine era at a major university hospital was nearly constant for each month of age during the first year of life, revealing an unexpectedly high incidence of symptomatic rotavirus disease in infants <3 months old. A sizeable fraction of cases occurred in children too young to have been vaccinated according to current recommendations.
PMCID: PMC2908071  PMID: 20540748
10.  Emergence of unusual human rotavirus strains in Salento, Italy, during 2006–2007 
In recent years, rotavirus genotyping by RT-PCR has provided valuable information about the diversity of rotaviruses (RV) circulating throughout the world.
The purpose of the present study was to monitor the prevalence of the different G and P genotypes of rotaviruses circulating in Salento and detect any uncommon or novel types.
During the period from January 2006 to December 2007, a total of 243 rotavirus positive stool samples were collected from children with diarrhoea admitted to four Hospitals in the province of Lecce (Copertino, Galatina, Gallipoli and Tricase).
All the specimens were tested for RV by real time PCR and genotyped for VP7 (G-type) and VP4 (P-type) gene by reverse transcription (RT) and multiplex PCR using different type specific primers.
In course of this study we identified 4 common G&P combinations viz. G2P[8], G1P[8], G2P[4] and G9P[8] amongst 59.8% of the typeable rotavirus positives.
Rotavirus G2P[8] was recognized as the most widespread genotype during the sentinel-based survey in Salento.
The detection of other novel and unusual strains, such as G2P[10], G4P[10], G8P[4], G9P[11] and G10P[8] is noteworthy.
Furthermore, a significant number of mixed infections were observed during the survey period but G3P[8] rotaviruses were not detected.
This study highlights the genetic diversity among rotaviruses isolated from children in Salento and the emergence of some novel strains. Therefore, it is highly essential to continuously monitor for these strains so as to assess the impact of vaccines on RV strains circulating in Salento and understand the effect of strain variation on efficacy of presently available vaccines.
PMCID: PMC2676288  PMID: 19368717
11.  1998-1999 Rotavirus Seasons in Juiz de Fora, Minas Gerais, Brazil: Detection of an Unusual G3P[4] Epidemic Strain 
Journal of Clinical Microbiology  2002;40(8):2837-2842.
An epidemiologic survey on the rotavirus strains causing gastroenteritis in young children was conducted in Juiz de Fora, Minas Gerais, in Southern Brazil during two consecutive seasons. Rotavirus was detected in 94 of the 1,056 fecal specimens collected from January 1998 to December 1999. Among the 13 discernible long electrophoretic profiles found, one was highly prevalent (73.4%) and represented the rotavirus strain responsible for the May-August winter epidemic outbreak of 1998, as clearly shown in a three-dimensional graph. This epidemic strain, designated JF98, was characterized as subgroup II and genotype G3P[4] by the original reverse transcription-PCR typing assays. Besides the unusual combination of G and P types, this G3 strain lacked reactivity with anti-G3-specific monoclonal antibodies and presented an uncommon pattern upon digestion of its cDNA-copied VP7 gene with the BstYI restriction enzyme. Strain JF98 affected primarily 6- to 24-month-old children and accounted for 85.5% of the severe rotavirus-associated dehydrating diarrhea cases that required hospitalization. As in our previous studies in neighboring Rio de Janeiro and São Paulo, a remarkably large proportion (44%) of mixed infections was detected, generating a complex set of circulating strains in the community, represented by the many distinct electropherotypes. Other common human types were detected as minor strains in single or in mixed infections, including the JF98 strain. Those were types G1, G4, G8, G9, P[8], and P[6], but not G2 or G5. One specimen contained a mixture of group A and C rotaviruses.
PMCID: PMC120661  PMID: 12149339
12.  Molecular Epidemiology of Rotavirus Strains Circulating among Children with Gastroenteritis in Iran 
Iranian Journal of Pediatrics  2012;22(1):63-69.
This study was conducted to evaluate the prevalence of rotavirus disease and to investigate the genotypes of rotavirus strains causing acute gastroenteritis among children aged <5 years old in Marvdasht, Iran.
One hundred and forty-one children, aged 1 month to 5 years, afflicted with severe diarrhea were enrolled during January 2007 to December 2008. Their stool samples were studied with enzyme immunoassays (EIA) for group A rotaviruses. Rotavirus-positive specimens were genotyped by the Nested RT-PCR using different types of specific primers.
Out of total collected samples rotavirus infection was detected in 40 (28.37%). Of the rotavirus episodes, 72.91% occurred during the first 2 years of life (P=0.038). The highest prevalence of infection was identified in summer (52.50%) and the lowest in winter (7.50%). The most common clinical features included diarrhea (96.25%), vomiting (82.50%) and fever (45.0%). Mixed genotypes were the predominant G type (60.0%), followed by non-typeable (12.50%), G2 (12.50%), G4 (10.0%) and G1 (5.0%) genotypes. G3/G8 mixed infection is the first of these rotavirus genotypes to be reported in Iran.
Regarding high frequency of rotavirus infection, continuous surveillance is needed to inform diarrhea prevention programs as well as to provide information about the occurrence of new rotavirus strains. This will assist policy makers in decision making on rotavirus vaccine introduction.
PMCID: PMC3448217  PMID: 23056861
Rotavirus; Gastroenteritis; Genotyping; Children; Epidemiology
13.  Epidemiological and clinical features of rotavirus among children younger than 5 years of age hospitalized with acute gastroenteritis in Northern Italy 
BMC Infectious Diseases  2010;10:218.
Rotavirus is the major cause of acute gastroenteritis and severe dehydrating diarrhea in young children.
To estimate the proportion of hospital admissions for rotavirus acute gastroenteritis and identify the circulating G and P genotypes among children under five years of age, we conducted a prospective observational study from January to December 2008, recruiting children consecutively admitted to six hospitals in Milan and nearby towns in northern Italy. Typing was done on stool samples by reverse transcriptase polymerase chain reaction amplification.
Of the 521 stool samples from children with acute gastroenteritis, 34.9% (95%CI, 30.8 to 39.2%) were rotavirus-positive. Two thirds (67.6%) were under two years of age, and 13.2% were under six months. The predominant G type was G1 (40.7%), followed by G9 (22.5%), G2 (13.2%), G3 (5.5%), G4 (3.8%) and G10 (1.6%). Twenty-one (11.7%) mixed-G infections were identified: G1+G10 (8.8%); G1+G9 (1.6%); and G2+G10 (1.2%). Only P[8] (67.6%) and P[4] (12.6%) types were P genotyped. The predominant single G/P combination was G1P[8] (39.7%), followed by G9P[8] (25.3%), G2P[4] (14.3%), and G3P[8] (4.1%). All G-mixed types combined with P[8].
These findings show an high prevalence of rotavirus infections among children admitted to hospital for acute gastroenteritis caused by different rotavirus strains circulating in the area studied.
PMCID: PMC2918608  PMID: 20649961
14.  Molecular characterization of rotavirus isolates from select Canadian pediatric hospitals 
BMC Infectious Diseases  2012;12:306.
We report the first multi-site rotavirus genotype analysis in Canada. Prior to this study, there was a dearth of rotavirus G and P genotyping data in Canada. Publically funded universal rotavirus vaccination in Canada started in 2011 and has been introduced by four provinces to date. Uptake of rotavirus vaccines in Canada prior to 2012 has been very limited. The aim of this study was to describe the genotypes of rotavirus strains circulating in Canada prior to widespread implementation of rotavirus vaccine by genotyping samples collected from selected paediatric hospitals. Secondly we identified rotavirus strains that differed genetically from those included in the vaccines and which could affect vaccine effectiveness.
Stool specimens were collected by opportunity sampling of children with gastroenteritis who presented to emergency departments. Samples were genotyped for G (VP7) genotypes and P (VP4) genotypes by hemi-nested multiplex PCR methods. Phylogenetic analysis was carried out on Canadian G9 strains to investigate their relationship to G9 strains that have circulated in other regions of the world.
348 samples were collected, of which 259 samples were rotavirus positive and genotyped. There were 34 rotavirus antigen immunoassay negative samples genotyped using PCR-based methods. Over the four rotavirus seasons, 174 samples were G1P[8], 45 were G3P[8], 22 were G2P[4], 13 were G9P[8], 3 were G4P[8] and 2 were G9P[4]. Sequence analysis showed that all Canadian G9 isolates are within lineage III.
Although a limited number of samples were obtained from a median of 4 centres during the 4 years of the study, it appears that currently approved rotavirus vaccines are well matched to the rotavirus genotypes identified at these hospitals. Further surveillance to monitor the emergence of rotavirus genotypes in Canada is warranted.
PMCID: PMC3519651  PMID: 23153184
15.  Annual incidence, serotype distribution, and genetic diversity of human astrovirus isolates from hospitalized children in Melbourne, Australia. 
Journal of Clinical Microbiology  1996;34(7):1750-1753.
The incidence of astrovirus infection in children under 5 years of age hospitalized for acute gastroenteritis in Melbourne, Australia, during 1995 was determined. Astrovirus was detected in 16 fecal specimens by Northern (RNA) dot blot analysis of RNA isolated from feces with an astrovirus-specific cDNA probe. The incidence of astrovirus infection was determined as 4.2% (16 of 378 total samples) compared with rates of 63.2, 3.7, and 4.2% for rotavirus, adenovirus, and all bacterial pathogens, respectively. Astrovirus was detected during the winter season and mainly in infants between 6 and 12 months of age. Serotyping of samples was carried out by reverse transcriptase PCR and direct sequencing of a 348-bp region of the capsid protein gene. Type 1 strains predominated (11 of 13 typeable samples), although type 4 isolates were also detected. Astrovirus was retrospectively identified in 13 fecal samples collected from hospitalized infants between 1980 and 1985 and shown to contain small viruses by electron microscopy. Type 1 isolates were again the most common, although a type 5 strain was also found. Comparative sequence analysis indicated that type 1 astroviruses exhibited up to 7% sequence divergence over a 15-year period; however, all mutations were silent. The incidence of astrovirus reported here indicates that the virus is a significant cause of severe diarrhea in young children. The genetic analysis also provides important molecular epidemiological information relevant to the development of preventative therapies.
PMCID: PMC229107  PMID: 8784582
16.  Molecular Characterization of Equine Rotavirus in Ireland▿  
Journal of Clinical Microbiology  2008;46(10):3346-3354.
Group A rotaviruses are important causative agents of severe, acute dehydrating diarrhea in foals. A total of 86 rotavirus-positive fecal samples, collected from diarrheic foals from 11 counties in three of the four provinces of Ireland, were obtained from the Irish Equine Centre in Kildare during a 7-year (1999 to 2005) passive surveillance study and were characterized molecularly to establish the VP7 (G type) and VP4 (P type) antigenic specificities. Fifty-eight samples (67.5%) were found to contain G3 viruses, while in 26 samples (30.2%) the rotaviruses were typed as G14 and in 2 samples (2.3%) there was a mixed infection, G3 plus G14. All samples except for two, which were untypeable, were characterized as P[12]. Fifty-eight percent of the samples were obtained from County Kildare, the center of the Irish horse industry, where an apparent shift from G3P[12] to G14P[12] was observed in 2003. By sequence analysis of the VP7 protein, the G3 Irish strains were shown to resemble viruses of the G3A subtype (H2-like) (97.1 to 100% amino acid [aa] identity), while the G14 Irish strains displayed 93.9 to 97.1% aa identity to other G14 viruses. In the VP8* fragment of the VP4 protein, the P[12] Irish viruses displayed high conservation (92.3 to 100% aa) with other equine P[12] viruses. Worldwide, G3P[12] and G14P[12] are the most prevalent equine rotavirus strains, and G3P[12] vaccines have been developed for prevention of rotavirus-associated diarrhea in foals. Investigations of the VP7/VP4 diversity of the circulating equine viruses and the dynamics of strain replacement are important for better assessing the efficacies of the vaccines.
PMCID: PMC2566120  PMID: 18716232
17.  Changing profile of rotavirus genotypes in Bangladesh, 2006–2012 
BMC Infectious Diseases  2013;13:320.
Rotavirus is the leading cause of severe diarrhea in infants and young children worldwide including Bangladesh. Unlike what was seen in high-income countries, the licensed rotavirus vaccines did not show high efficacy in Bangladeshi trials. We assessed rotavirus prevalence and genotypes in Bangladesh over six-year period to provide baseline information on the rotavirus burden and changing profile in the country.
This study was conducted from June 2006 to May 2012 in Matlab, Bangladesh. Group A rotaviruses were detected in stools collected from diarrhea patients by ELISA and genotyped using multiplex reverse transcription PCR followed by nucleotide sequencing.
Of the 9678 stool samples, 20.3% were positive for rotavirus. The most predominant genotype was G1P[8] (22.4%), followed by G9P[8] (20.8%), G2P[4] (16.9%) and G12P[8] (10.4%). Mixed infections were detected in 14.2% of the samples. Emergence of an unusual strain, G9P[4] was documented during 2011–12. Several amino acid mismatches in the antigenic epitopes of VP7 and VP4 between Bangladeshi and the vaccine strains were identified.
Our study provides important information on rotavirus genotypes that should be considered for the selection and introduction of rotavirus vaccines in Bangladesh.
PMCID: PMC3723515  PMID: 23855423
Rotavirus; Diarrhea; Vaccine; Bangladesh; Genotype; Epitope
18.  The Emergence of Rotavirus G12 and the Prevalence of Enteric Viruses in Hospitalized Pediatric Diarrheal Patients in Southern Vietnam 
Diarrhea is a major cause of childhood morbidity and mortality in developing countries, and the majority of infections are of viral etiology. We aimed to compare the etiological prevalence of the major enteric viruses in an urban and a rural setting in southern Vietnam. We simultaneously screened fecal specimens from 362 children in Ho Chi Minh City and Dong Thap province that were hospitalized with acute diarrhea over a 1-month-long period for four viral gastrointestinal pathogens. Rotavirus was the most common pathogen identified, but there was a differential prevalence of rotavirus and norovirus between the urban and rural locations. Furthermore, rotavirus genotyping and phylogenetic analysis again differentiated the genotypes by the sampling location. Our data show a disproportional distribution of enteric viral pathogens in urban and rural locations, and we provide evidence of continual importation of new rotavirus strains into southern Vietnam and report the emergence of rotavirus genotype G12.
PMCID: PMC3183790  PMID: 21976585
19.  Prevalence and Molecular Genotyping of Group A Rotaviruses in Iranian Children 
Rotavirus is the leading cause of acute gastroenteritis in worldwide young children. Effective vaccines to prevent rotavirus infection are currently available, although their clinical use is still limited, and rotavirus still causes many episodes of infantile gastroenteritis, mainly during the winter season. The aim of this study was to evaluate the prevalence of rotavirus infection in children aged <5-years-old who were hospitalised for gastroenteritis. One hundred and sixty-three stool samples from hospitalised children (<5-years-old) complicated with severe diarrhoea, in two hospitals in Jahrom City, Iran were collected from 2009 to 2010. Antigenic prevalence of rotavirus group A was distinguished by enzyme immunoassay. The antigen of group A rotavirus was diagnosed by EIA in 75 of 163 collected samples. The genotype of EIA-positive samples was determined by nested RT-PCR. The frequency of rotavirus genotypes G1, G2, G3, G4 and G9 was 17.33, 13.34, 2.67, 30.66 and 2.67 %, respectively. Also, the frequency of mixed and non-typable genotypes was detected in 2.67 and 30.66 %, respectively. G1/G8 mixed infection was the first of these rotavirus genotypes to be reported in Iran. Detection of high prevalence of group A rotavirus infection in hospitalised children with diarrhoea, and determination of circulating rotavirus genotypes in this region of Iran, provide useful data for formulating effective vaccines; especially for infants less than 5-years-old.
PMCID: PMC3550810  PMID: 23729998
Rotavirus; Prevalence; Molecular genotyping; RT-PCR
20.  Epidemiology and Genetic Diversity of Rotavirus Strains in Children with Acute Gastroenteritis in Lahore, Pakistan 
PLoS ONE  2013;8(6):e67998.
Pakistan harbors high disease burden of gastro-enteric infections with majority of these caused by rotavirus. Unfortunately, lack of proper surveillance programs and laboratory facilities have resulted in scarcity of available data on rotavirus associated disease burden and epidemiological information in the country. We investigated 1306 stool samples collected over two years (2008–2009) from hospitalized children under 5 years of age for the presence of rotavirus strains and its genotypic diversity in Lahore. The prevalence rate during 2008 and 2009 was found to be 34% (n = 447 out of 1306). No significant difference was found between different age groups positive for rotavirus (p>0.05). A subset of EIA positive samples was further screened for rotavirus RNA through RT-PCR and 44 (49.43%) samples, out of total 89 EIA positive samples, were found positive. G and P type prevalence was found as follows: G1P [4] = 3(6.81%); G1P [6] = 9(20.45%); G1P [8] = 1(2.27%); G2P [4] = 21(47.72%); G2P [8] = 1(2.27%); G9P [4] = 1(2.27%); G9P [6] = 1(2.27%) and G9P [8] = 7(15.90%). Phylogenetic analysis revealed that the VP7 and VP4 sequences clustered closely with the previously detected strains in the country as well as Belgian rotaviruses. Antigenic characterization was performed by analyzing major epitopes in the immunodominant VP7 and VP4 gene segments. Although the neutralization conferring motifs were found variable between the Pakistani strains and the two recommended vaccines strains (Rotarix™ and RotaTeq™), we validate the use of rotavirus vaccine in Pakistan based on the proven and recognized vaccine efficacy across the globe. Our findings constitute the first report on rotavirus’ genotype diversity, their phylogenetic relatedness and epidemiology during the pre-vaccination era in Lahore, Pakistan and support the immediate introduction of rotavirus vaccine in the routine immunization program of the country.
PMCID: PMC3692488  PMID: 23825693
21.  Neonatal Infection with G10P[11] Rotavirus Did Not Confer Protection against Subsequent Rotavirus Infection in a Community Cohort in Vellore, South India 
The Journal of infectious diseases  2007;195(5):625-632.
Various observational studies have suggested that neonatal rotavirus infection confers protection against diarrhea due to subsequent rotavirus infection. We examined the incidence of rotavirus infection and diarrhea during the first 2 years of life among children infected with the G10P[11] rotavirus strain during the neonatal period and those not infected with rotavirus.
Children were recruited at birth and were followed up at least twice weekly. Stool samples, collected every 2 weeks for surveillance and at each episode of diarrhea, were screened by enzyme-linked immunosorbent assay and were genotyped by polymerase chain reaction.
Among 33 children infected neonatally with G10P[11] and 300 children not infected with rotavirus, there was no significant difference in the rates of rotavirus-positive diarrhea (rate ratio [RR], 1.05 [95% confidence interval {CI}, 0.61–1.79]), moderate or severe rotavirus-positive diarrhea (RR, 1.42 [95% CI, 0.73–2.78]), or asymptomatic rotavirus shedding (RR, 1.25 [95% CI, 0.85–1.83]).
Neonatal G10P[11] infection with a strain resembling a vaccine candidate did not confer protection against subsequent rotavirus infection or diarrhea of any severity in this setting.
PMCID: PMC2483790  PMID: 17262703
22.  How dangerous is food allergy in childhood? The incidence of severe and fatal allergic reactions across the UK and Ireland 
Archives of Disease in Childhood  2002;86(4):236-239.
Aims: To discover the incidence of fatal and severe allergic reactions to food in a large population of children.
Methods: A retrospective search for fatalities in children 0–15 years from 1990 to February 1998, primarily of death certification at offices of national statistics. A prospective survey of fatal and severe reactions from March 1998 to February 2000, primarily through the British Paediatric Surveillance Unit. Main outcome measures were deaths and severe reactions. A case was deemed severe if one or more of the following criteria was met: cardiorespiratory arrest; need for inotropic support; fluid bolus >20 ml/kg; more than one dose of epinephrine; more than one dose of nebulised bronchodilator. A case was deemed near fatal if intubation was necessary.
Results: The UK under 16 population is 13 million. Over the past 10 years, eight children died (incidence of 0.006 deaths per 100 000 children 0–15 years per year). Milk caused four of the deaths. No child under 13 died from peanut allergy. Two children died despite receiving early epinephrine before admission to hospital; one child with a mild food allergic reaction died from epinephrine overdose. Over the past two years, there were six near fatal reactions (none caused by peanut) and 49 severe ones (10 caused by peanut), yielding incidences of 0.02 and 0.19 per 100 000 children 0–15 years per year respectively. Coexisting asthma is more strongly associated with a severe reaction than the severity of previous reactions.
Conclusions: If 5% of the child population have food allergy, the risk that a food allergic child will die from a food allergic reaction is about 1 in 800 000 per year. The food allergic child with asthma may be at higher risk. Prescribing an epinephrine autoinjector requires a careful balance of advantages and disadvantages.
PMCID: PMC1719140  PMID: 11919093
23.  Phylogenetic Analysis of the Rotavirus Genotypes Originated from Children < 5 Years of Age in 16 Cities in South Korea, between 2000 and 2004 
The purpose of this study was to examine the diversity of the G and P types of human rotavirus strains isolated in South Korea during 2000 to 2004.
We selected 38 Group A rotavirus isolates among 652 fecal samples, which were collected from infants and children < 5 years of age with acute gastroenteritis or diarrhea admitted in 8 hospitals representative of five provinces of South Korea between 2000 and 2004. Rotavirus P- and G-genotypes were determined by nucleotide sequencing and phylogenetic analysis was performed.
One G1P[4] consisted G1-Id-P[4]-V; one G1P[6] consisted G1-Id-P[6]-Ia; nine G1P[8] consisted G1-Ib-P[8]-Ia (n=3), G1-Ic-P[8]-Ia (n=1), and G1-Id-P[8]-Ia (n=5); 13 G2P[4] consisted G2-V-P[4]-V; two G3P[4] consisted G3-IIId-P[4]-V; five G3P[8] consisted G3-IIId-P[8]-Ia; four G4P[6] consisted G4-Ie-P[6]-Ia; two G4P[8] consisted G4-Ie-P[8]-II; one G9P[6] consisted G9-III-P[6]-Ia.
A considerable amount of rotavirus genotypic diversity was detected in South Korea from 2000 to 2004. These findings are important to develop the effective vaccines and to undertake epidemiologic studies.
PMCID: PMC3738675  PMID: 24159485
human rotavirus vaccine; phylogenetic analysis; rotavirus; South Korea; viral gastroenteritis
24.  Rotavirus G and P Genotypes in Rural Ghana 
Journal of Clinical Microbiology  2001;39(5):1981-1984.
An epidemiological study of rotavirus infection was conducted on specimens collected from patients with gastroenteritis and domiciled in the rural Upper Eastern Region of Ghana during 1998. Fifty isolates, randomly selected from 165 human group A rotavirus-positive samples, were G and P characterized by a reverse transcription (RT)-PCR assay using a seminested multiplex method. Rotaviruses of the G3 genotype were found to be the predominant strain (78%), followed by G2 (14%) and G1 (2%). Mixed infections, as shown by combinations of G3 and G2 (4%) and G3 and G1 (2%), were also observed. P typing showed P[4] (72.34%) to be the prevalent strain, followed by P[6] (21.3%), P[8] (2.13%), and a combination of P[4] and P[6] (4.3%).
PMCID: PMC88064  PMID: 11326029
25.  Rapid Changes in Rotaviral Genotypes in Ecuador 
Journal of medical virology  2009;81(12):2109-2113.
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.
PMCID: PMC2882630  PMID: 19856474
rotavirus; genotype analysis; Ecuador; community study

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