in 1994 , the metropolitan area environmental health service ( health service ) began a gastroenteritis outbreak surveillance program in the centers .
this study was to determine the role of hucvs as a cause of gastroenteritis outbreaks from june 1 , 2000 , to january 30 , 2003 , in santiago , chile , by using recently improved antigen and genome detection assays , and to characterize genetically the circulating strains .
sentinel sites were instructed to report gastroenteritis outbreaks 48 hours after detecting the sentinel case .
a health service epidemiologist would initiate an investigation and make home visits to identify all persons possibly involved in the outbreak .
stools samples for pathogen detection were collected during home visits from affected persons and were cultured for salmonella , shigella , campylobacter , and vibrio spp . , according to standard techniques using selective media ( 10 ) .
enteropathogenic escherichia coli , enterotoxigenic e. coli , and enterohemorrhagic e. coli were studied by multiplex polymerase chain reaction ( 11 ) and enzyme - linked immunosorbent assay ( elisa ) .
rotavirus and enteric adenoviruses were detected by elisa or by commercial kits ( sas rota test , sa scientific inc .
, san antonio , tx , usa ; premier adenoclone , meridian diagnostics inc . ,
cincinnati , oh , usa ; 40/41 adeno - strip , coris bioconcept , gembloux , belgium ) and parasites were detected by burrows technique .
all samples were tested for hucv by a novel elisa specific for noroviruses based on pools of sera obtained from rabbits and guinea pigs hyperimmunized with a total of 9 different norovirus capsids ( 12 ) and by reverse transcription  polymerase chain reaction ( rt - pcr ) targeting conserved sequences in the polymerase region of hucvs ( 9 ) .
primers used for rt - pcr were 289 ( rt)/290 ( pcr ) or a pool of degenerate primers of last generation , 289hi for rt and 290hijk for pcr , that detect norovirus and sapovirus ( 13,14 ) .
rt - pcr products were cloned by using pgem - t easy vector system ( promega , madison , wi , usa ) .
the 327-base nucleotide sequences that encode for the polymerase dependent rna were aligned by using omiga 2.0 ( oxford molecular , madison , wi , usa ) software and compared with 21 prototype sequences retrieved using blast searches from the genbank database .
phylogenetic distances were calculated by kimura 2-parameter method and a phylogenetic tree was plotted by the neighbor - joining method using mega , version 2.1 ( 15 ) .
during the 30-month study , a total of 82 outbreaks affecting 100 persons in the santiago metropolitan area were reported properly to the health service and investigated . in each outbreak , a rectal swab from 1 person was collected for microbial studies . in each of 55 outbreaks ,
1 stool sample was collected for virus studies , and in each of 31 outbreaks , 1 stool sample was collected for parasite studies .
enteric microbial pathogens were isolated in samples from 1 person in 32% of the 82 outbreaks , and potentially pathogenic parasites were isolated in 6 ( 19% ) of 31 outbreaks ( table 1 ) .
a total of 175 samples from 55 outbreaks were obtained for viral detection , of which 47 ( 27% ) from 25 ( 45% ) outbreaks were positive for hucv by using 1 method .
hucv outbreaks affected 99 persons with a median of 5 persons ( table 1 ) . in 16 outbreaks , 2 persons were positive by using elisa or rt - pcr ; in 9 outbreaks , 1 person was positive by 1 method .
overall , 20% of the outbreaks were detected only by elisa , 24% only by rt - pcr , and 56% by both techniques .
* an outbreak was associated with a given pathogen if 1 sample was positive . 
bacteria isolated included : enteropathogenic escherichia coli ( epec ) ( 2 ) , enterotoxigenic e. coli ( etec ) ( 3 ) , enterohemorrhagic e. coli ( 3 ) , epec + etec ( 1 ) , salmonella sp ( 12 ) , shigella sp ( 2 ) , staphylococcus aureus ( 3 ) . 
january 110 , 2003 .   in 1 outbreak , etec and epec and in another , shiga toxin 
in 1 additional outbreak the only pathogens simultaneously detected in 1 patient were rotavirus and adenovirus by enzyme - linked immunosorbent assay .
most hucv outbreaks occurred in the home , with outbreaks in childcare centers and schools occurring next most frequently ; only a small fraction occurred in restaurants .
the most commonly implicated food products were seafood , including raw oysters and clams ( table 2 ) . among a total of 1,137 persons exposed in the 25 hucv outbreaks , 283 ( 25% ) had typical acute gastroenteritis symptoms .
thirty - nine percent of the cases occurred in children < 5 years of age , 28% occurred in children 514 years of age , 27% occurred in adolescents and adults 1560 years of age , and 4% occurred in adults > 60 years of age .
most commonly reported symptoms were diarrhea ( 86% ) , vomiting ( 36% ) , and fever ( 16% ) . *
hucv amplicons from 13 outbreaks evaluated belonged to the norovirus genus , including 8 gii , 2 gi , and 3 in a potentially novel genogroup .
the 3 new strains differed > 40% in nucleotide identity from all prototype strains compared ( figure ) .
bootstrap analysis based upon 1,000 generated trees yielded a node for the potentially novel genogroup in 100% of the trees .
two of the outbreaks caused by this potentially novel genogroup occurred during the same month , while the third occurred a year later .
one of the genetic clusters , represented by strain 028/10 - 2001 , was closely related with a distance of 0.11 to saitama virus ( saiu1 , accession no .
ab039775 ) , a japanese strain found in 1998 in a child with acute gastroenteritis .
the 2 other genetic clusters are proposed as novel genetic clusters and include strains ( i ) o55/5 - 2002 , o64/10 - 2002 , o62/9 - 2002 , o71/11 - 2002 , o78/11 - 2002 , and ( ii ) o77/11 - 2002 , o85/1 - 2003 ( figure ) .
the first cluster has 2 independent nodes with a distance of 0.19 to 0.28 from saiu1 , the second cluster is represented by 2 strains with a distance of 0.18 and 0.19 from saiu1 , respectively .
phylogenetic tree of noroviruses based on the 327-base region of the 3  end of the open reading frame 1 using 13 novel sequences designated according to outbreak number / month  year ( example : o55/52002 ) , and 21 sequences of norwalk - like virus strains representative of the currently identified genogroups , designated according to genbank accession number .
comparative strains include : norwalk virus ( m87661 ) , saitamau1 ( ab039775 ) , saitama u201 ( ab039782 ) , wug1 ( ab081723 ) , schreier ( af093797 ) , camberwell ( af145896 ) , fort lauderdale ( af414426 ) , saint cloud ( af414427 ) , jena ( aj011099 ) , maryland ( ay032605 ) , murine nv ( ay228235 ) , southampton ( l07418 ) , oth25 ( l23830 ) , snow mountain ( l23831 ) , toronto ( u02030 ) , desert shield virus ( u04469 ) , hawaii ( u07611 ) , mexico ( u22498 ) , bristol ( x76716 ) , melksham ( x81879 ) , and lorsdale ( x86557 ) .
bootstrap values based on 1,000 generated trees are displayed at the nodes ( values > 60% are shown ) .
hucvs were associated with almost half of 55 fully evaluated gastroenteritis outbreaks in santiago , chile , and were more common than outbreak - associated enteric bacterial pathogens such as salmonella sp . and diarrheogenic e. coli . to our knowledge
, this is the first prospective , active surveillance for gastroenteritis outbreaks in latin america that included a thorough search for hucvs .
publications from the region have described high seroprevalence for these viruses ( 3,16 ) and have reported isolated outbreaks affecting children and adults ( 3,8 ) .
hucv - associated outbreaks mostly affected children that ate seafood in homes ; other implicated sources included meat products and vegetables .
the reported outbreaks in this study reflect the tip of the iceberg ; only 10% of all reported outbreaks could be studied because of capacity and resources for prompt reporting and investigation .
this study should stimulate efforts for appropriate outbreak investigation in developing regions where food products safety is important for the health of the population , tourism , and international commerce .
genogroup ii strains dominated , as in other studies ( 36 ) , but only 1 of these strains fell into the same genetic cluster of a previously described strain , saitama virus ; in contrast , most strains grouped into 2 closely related new clusters .
in addition , 3 strains , 2 temporally related , likely belong to a new genogroup .
the circulation of genetically diverse strains indicates the need for further studies to understand the clinical and epidemiologic importance of such diversity .
 human caliciviruses caused 45% of 55 gastroenteritis outbreaks occurring in santiago , chile , during 20002003 . 
 outbreaks affected 99 persons , occurred most commonly in the home , and were associated with seafood consumption . 
 thirteen outbreak strains sequenced were noroviruses , including 8 gii , 2 gi , and 3 belonging to a novel genogroup . 
The Study
Conclusions