from january 1998 through may 2005 , a total of 2,421 fecal specimens were collected from children < 10 years of age ( median age 2.3 years ) with acute diarrhea in rio de janeiro , brazil . of these ,
478 ( 19.7% ) specimens were collected from hospitalized children ( inpatients ) and 1,943 from outpatient children ( 341 [ 14.1% ] from the emergency department and 1,602 [ 66.2% ] from the walk - in clinic ) . of these samples ,
the median age was 12.5 months for rotavirus - positive patients and 12.2 months for adenovirus - positive patients .
overall , of the hospitalized , emergency department , and walk - in clinic patients , 11.7% , 6.2% , and 10.0% , respectively , had samples positive for rotavirus , and 4.8% , 4.1% , and 4.4% , respectively , had samples positive for adenovirus .
yersinia enterocolitica , campylobacter spp . , and shigella spp . were found in 8% of the specimens .
, 1 rotavirus and salmonella spp . , and 1 rotavirus and e. coli ) .
we selected 289 specimens that represented a random subset of samples that had prior negative results for rotavirus and enteric adenovirus .
of these 289 , 117 were collected from inpatients and 172 from outpatients ( 89 emergency department and 83 walk - in clinic ) .
suspensions of stool ( 10% ) were prepared in diethylpyrocarbonate - treated water and vertrel xf ( miller - stephenson , sylmar , ca , usa ) and clarified by centrifugation at 2,100 g for 10 min .
we used 200 l of suspension for rna extraction with the nuclisens extraction kit ( organon tekninka , durham , nc , usa ) according to the manufacturer s instructions .
the rna was eluted in 50 l of elution buffer and stored at 70c until use .
a total of 240 samples were tested for norovirus rna by light cycler pcr that used primers and probes for orf1/orf2 junction region specific for norovirus gi and gii , as described ( 3,12 ) , and by the light cycler rna amplification kit hybridization probes ( roche , basel , switzerland ) .
the 49 remaining samples were tested only by conventional rt - pcr , as described ( 5 ) .
conventional rt - pcr was performed with the qiagen onestep rt - pcr kit ( qiagen , valencia , ca , usa ) .
the rna samples were subjected to 1 cycle of reverse transcription ( 42c , 10 min ) followed by 5 min at 95c .
pcr was performed for 40 cycles , each consisting of 1 min at 94c , 1 min at 40c ( for gi detection ) or 1 min at 44c for ( for gii detection ) , 1 min at 72c , and a final extension cycle of 10 min at 72c .
we selected 6 samples that were positive by real - time light cycler pcr ( 2 gi and 4 gii ) for analysis by conventional rt - pcr with specific primers in capsid region d of norovirus gi and gii , as described above .
the amplified cdna samples were purified from the gel by using qiaquick gel extraction kit ( qiagen ) , and the sequences were determined with the bigdye terminator cycle sequencing kit and the abi prism 3100 automated dna sequencer ( applied biosystems , foster city , ca , usa ) by using the same primers as used for the conventional rt - pcr .
the nucleotide sequences of the amplicons were aligned with corresponding sequences of selected norovirus strains available in the genbank database and analyzed by using the clustal v algorithm of the megalign program in the dnastar software package ( madison , wi , usa ) .
dq496212 , dq496213 , dq496214 , dq496215 , dq496216 , and dq496217 . of the 289 fecal specimens tested , 42 ( 14.5% ) were positive for norovirus : 36 ( 15% ; n = 240 ) by real time light cycler pcr and 6 ( 12.2% ; n = 49 ) by conventional rt - pcr .
these percentages correspond only to single infections because we did not test samples already known to be positive for other pathogens such as rotavirus and adenovirus .
positive samples and genogroups varied by year with no obvious yearly pattern ( table 1 ) .
the winter vomiting disease ,  we detected infection throughout the year , with no seasonal pattern ( figure ) .
norovirus infections were equally common among outpatients and inpatients . among 117 inpatients , 18 ( 15.4% ) had positive norovirus test results compared with 24 ( 14% ) of 172 outpatients ( 11 emergency department and 13 walk - in clinic ) .
although the disease caused by norovirus is described as mild ( diarrhea , vomiting , abdominal pain , and fever ) and generally does not lead to hospitalization ( 13,14 ) , of 42 norovirus - infected children , 29 ( 69% ) were either hospitalized or received medical care in the emergency department , which suggests that they had a more severe illness . only 13 ( 31% ) of the 42 norovirus - infected children attended the walk - in clinic , which suggests that they had mild disease ( table 2 ) .
other than diarrhea , fever was the most common symptom among the 42 norovirus - positive patients in this study , reported for 11 ( 26.2% ) patients . vomiting only was described for 8 ( 19.0% ) ; vomiting and fever was described for 6 ( 14.3% ) . no mixed infection with bacteria
seasonal distribution of norovirus ( nov ) infections in rio de janeiro , brazil , 19982005 .
although norovirus belonging to genogroup gii is considered the most prevalent strain worldwide ( 79,11,15 ) , we found no important difference in the prevalence of the 2 genogroups detected in our study .
overall , 20 ( 48% ) of the 42 samples were identified as genogroup gi and 22 ( 52% ) as gii ( table 1 ) .
no statistically significant difference in the prevalence of gi and gii was observed between inpatients and outpatients .
our study documents that noroviruses are a common cause of acute gastroenteritis in children who are inpatients or outpatients in brazil and are likely second only to rotavirus as a cause of severe childhood diarrhea .
nonetheless , it documents how common norovirus infections may be and indicates that further study will be necessary to assess their role among brazilian children , to understand the epidemiology of the disease , and to seek evidence of immunity in children , which might encourage development of a vaccine .
 during 19982005 , we analyzed stool samples from 289 children in rio de janeiro to detect and genotype norovirus strains . 
 previous tests showed all samples to be negative for rotavirus and adenovirus . 
 of 42 ( 14.5% ) norovirus - positive specimens , 20 ( 47.6% ) were identified as genogroup gi and 22 ( 52.3% ) as gii . 