most of escherichia coli isolated from stool are nonpathogenic . during the last decade , many researchers reported isolation of pathogenic strains as causes of large food  borne outbreaks in developed counties .
from 5 main pathogenic e. coli strains , shiga toxin - producing e. coli ( stec ) that produces shiga - like toxins received more attention .
although e. coli o157:h7 is the most common serotype that produces this toxin , non o157:h7 has also been reported to cause infection in children with no significant differences in severity of illness .
cattle , in which the bacterium inhabits intestinal flora , is the natural reservoir of stec . the infection is mostly acquired by human beings through the ingestion of contaminated water and under - cooked food although human to human transmission has been described . mortality and morbidity of diarheal disease in stec infection is an important health care problem especially in young children
. meanwhile , hemolytic uremic syndrome ( hus ) , acute renal failure and end stage renal disease are other complications of this infection . 

stx1 and stx2 , enterotoxins which are produced by stec , are structurally related to the toxin produced by shigella dysenteriae .
shiga - toxins have a and b subunits , the latter binds to the surface of enterocytes , polymerizes in plasma membrane , and supplying a route for the other subunit to enter the cytoplasm .
being proteolytically activated in cytoplasm , the a subunit , irreversibly inhibits protein synthesis by inactivating the ribosome , and kills the cell . carried to kidneys by blood stream ,
shiga - toxins cause endothelial damage in glomerular capillaries , which accompanied by platelet aggregation and fibrin deposition , finally results in renal failure .
there are several studies that determined the frequency of infection by stec in diarrheal patients and evidence show that stec can be a major problem in developed and developing countries , but there are only few published data from iran .
stec can be diagnosed by different methods ; stool culture on sorbitol - macconkey agar ( smac ) mostly used for detection of e. coli o157 .
enzyme immunoassay for shiga toxins 1 and 2 and molecular assay for stx genes are other methods . here
we report the frequency of stec infection with pcr in patients with diarrhea referred to children 's medical center in tehran .
all patients with diarrhea or dysentery admitted to children 's medical center from october 1 , 2008 , to september 1 , 2009 , were enrolled in this cross sectional study .
stool samples were collected and cultured on emb ( eosin - methylene blue ) agar at 37c .
after 24 hours , recognizable colonies with metallic shine examined biochemically for isolating e. coli strains .
indole ( + ) , methyl red ( + ) , simon citrate ( - ) , urea hydrolase ( - ) , lysine decarboxylase ( + ) , onpg ( + ) and tsi [ alk / a , h2s(- ) ,
gas(- ) ] colonies were inoculated into two cary - blair transport media and preserved there at 27c for 24 hours .
one of the two cary - blair media was stored at -20 c for further study , and the second one was inseminated to muller agar and incubated at 37c for 24 hours before extraction .
dna from biochemically confirmed isolates was extracted with method described by dhanashree and mallya with minor modifications .
briefly , a loopful of bacterial colonies , from muller agar , admixed with 200 l sterile distilled water and then placed in a water bath at 97c for 15 minutes .
the samples were centrifuged for 5 minutes at 8000 g , and then the supernatant was transferred to a fresh tube and stored at 20c for polymerase chain reaction . for each reaction , master mix contained 10x pcr buffer ( bioflux , tokyo , japan ) , 2.5 millimolar of mgcl2 , 1 millimolar of dntp ( bioflux , tokyo , japan ) , 2 iu of taq polymerase ( bioflux , tokyo , japan ) and 200 nanomolar of each primer ( cinnagen , tehran , iran ) .
pcr amplification was performed by using a mastercycler gradient pcr machine ( eppendorf , netheler - hintz gmbh , hamburg , germany ) .
the duplex pcr conditions for stx1/stx2 involved denaturation of template dna for 4 minutes at 95c followed by 35 cycles of 30 seconds at 95c , 60 seconds at 55c and 60 seconds at 72c , final extension period was 7 minutes at 72c . after pcr completion , gel electrophoresis was preformed and the lengths of amplicons were compared with predetermined ladder ( fig .
c-:negative control , c+:positive control , l : ladder , 1 and 2 ( two samples , one of them stx1 and second stx2 positive ) primer sequences and length of amplification products the pcr products were detected by electrophoresis on 1% agarose gel at 100 volts for 60 minutes . in each run of electrophoresis , a dna ladder ( fermentas gene ruler 1 kb , cinnagen , tehran ,
gel was stained with ethidium bromide and then visualized on uv transilluminator , pcr products with 302 bp and 516 bp length were considered positive for stx1 and stx2 respectively ( fig .
all patients with diarrhea or dysentery admitted to children 's medical center from october 1 , 2008 , to september 1 , 2009 , were enrolled in this cross sectional study .
stool samples were collected and cultured on emb ( eosin - methylene blue ) agar at 37c .
after 24 hours , recognizable colonies with metallic shine examined biochemically for isolating e. coli strains .
indole ( + ) , methyl red ( + ) , simon citrate ( - ) , urea hydrolase ( - ) , lysine decarboxylase ( + ) , onpg ( + ) and tsi [ alk / a , h2s(- ) ,
gas(- ) ] colonies were inoculated into two cary - blair transport media and preserved there at 27c for 24 hours .
one of the two cary - blair media was stored at -20 c for further study , and the second one was inseminated to muller agar and incubated at 37c for 24 hours before extraction .
dna from biochemically confirmed isolates was extracted with method described by dhanashree and mallya with minor modifications .
briefly , a loopful of bacterial colonies , from muller agar , admixed with 200 l sterile distilled water and then placed in a water bath at 97c for 15 minutes .
the samples were centrifuged for 5 minutes at 8000 g , and then the supernatant was transferred to a fresh tube and stored at 20c for polymerase chain reaction .
for each reaction , master mix contained 10x pcr buffer ( bioflux , tokyo , japan ) , 2.5 millimolar of mgcl2 , 1 millimolar of dntp ( bioflux , tokyo , japan ) , 2 iu of taq polymerase ( bioflux , tokyo , japan ) and 200 nanomolar of each primer ( cinnagen , tehran , iran ) .
pcr amplification was performed by using a mastercycler gradient pcr machine ( eppendorf , netheler - hintz gmbh , hamburg , germany ) .
the duplex pcr conditions for stx1/stx2 involved denaturation of template dna for 4 minutes at 95c followed by 35 cycles of 30 seconds at 95c , 60 seconds at 55c and 60 seconds at 72c , final extension period was 7 minutes at 72c . after pcr completion , gel electrophoresis was preformed and the lengths of amplicons were compared with predetermined ladder ( fig .
c-:negative control , c+:positive control , l : ladder , 1 and 2 ( two samples , one of them stx1 and second stx2 positive ) primer sequences and length of amplification products
the pcr products were detected by electrophoresis on 1% agarose gel at 100 volts for 60 minutes . in each run of electrophoresis , a dna ladder ( fermentas gene ruler 1 kb , cinnagen , tehran , iran )
gel was stained with ethidium bromide and then visualized on uv transilluminator , pcr products with 302 bp and 516 bp length were considered positive for stx1 and stx2 respectively ( fig .
from all 6500 samples received , 967 e. coli strains were isolated and tested by pcr method .
twenty seven ( 2.8% ) cases were positive for stx1 or stx2 genes ( fig .
most common season for stx - positive e. coli was spring ( 17 out of 27 = 63% , p < 0.05 ) ( table 2 ) .
most of stx - positive samples had few wbc or rbc and none was bloody .
the details of results are summarized in table 3 . 
  seasonal prevalence in diarheal specimens with stx - positive
e.coli isolates and all e.coli isolates stool analysis findings of diarheal specimens with stx - positive e.coli isolates and all e.coli isolates stec : shiga toxin - producing e. coli 

its diagnosis from non pathogenic ecoli is important because of its ability to produce hemolytic uremic syndrome , a life - threatening disease in 15% of patients with 5% mortality rate .
treatment of infection with antibiotics is controversial because of increasing risk of hus . thus knowing the frequency , seasonal and epidemiologic pattern of stec in each population can guide clinicians in test ordering and management of diarrheic patients .
although e. coli o157:h7 is the most common serotype of stec 
 , non o157:h7 has also been reported to cause infection in children .
culture on specific medium ( smac ) and serological assay were used mostly for diagnosis . since non -o157 stec usually can not be recognized on smac agar , evaluation of stool samples for toxins ( proteins or gens )
so we decided to use a pcr method to determine the frequency of shiga toxin producing e. coli in diarrhea samples in our population and evaluate the relationship between stool exam findings and shiga toxin positivity . in our study 967 e. coli strains were isolated from diarrheal stool samples of children younger than 14 years from october 2008 to september 2009 and amplification of stx1 and stx2 genes using pcr method revealed that 2.8% of them were stec .
salmanzadeh - ahrabi , s et al in 2005 reported that stec was isolated in 15.5% of patients under 5 years of age with diarrhea . in another study in iran ,
alikhani et al in 2007 found that 8.7% of the children aged less than 10 years with diarrhea were infected with stec .
frequency infection with stec is different in other populations . in a study conducted in australia
another study in netherlands revealed the prevalence of stec in 3.8% of samples with macroscopic blood and 1.4% in diarrheal stool samples . in bennett - wood vr
we examined all specimens to find any association between stool exam findings and stec positivity . in this
regard we classified our data based on gross and microscopy such as stool color , pus , mucus , wbc and rbc .
there was no significant association between these findings and stec ; this indicates that all diarrheal stools should be examined for shiga toxin - producing e. coli if no stec is to be missed .
in contrast to our results appleman et al showed that 83% of the children with stec had blood in their stool samples .
we found that the rate of stec positive diarrhea was increased in the spring . in a study in germany ducker
c. et al reported an outbreak of shiga - toxin - producing escherichia coli ( ehec ) infections occurred from may to june and the great number of patients had hus .
there was no significant sex differences in the frequency of stec positive isolates in our study , although some studies found it higher in younger and female patients .
we did not determine the serotype of the isolates and could not compare our results with other studies to find if e. coli o157:h7 is the most prevalent stec in iran or not .
also , if we could have used smac agar for isolation , we could have differentiated e. coli o157:h7 from other e. coli strains .
the low frequency of stec in our population indicates that the stec and hus is not a major problem in our population but , using rapid and accurate methods for detection of it , is important because stec infection may lead to serious complications like hus .
further studies with greater sample size are needed for determining stec frequency and also frequency of shiga - toxin - associated hus .
 objectiveshiga toxin - producing e. coli ( stec ) is a pathogenic e. coli that may cause hemolytic uremic syndrome ( hus ) after diarrheal disease through shiga toxins . 
 management of the patients with stec infection is different from that of other diarrheal diseases due to increase in frequency of hus after antibiotic administration . 
 few studies were conducted in iran and epidemiology of stec remains obscure ; this necessitates examination of stools especially in young children for this bacterium.methodswe determined the frequency of stec in 947 e. coli strains isolated from diarrheal stools of children less than 14 years in tehran with conventional culture methods and multiplex - pcr via determining the stx1 and stx2 genes , between october 2008 and september 2009 . 
 we also evaluated the association between stool exam findings and presence of stec.findingstwenty seven ( 2.8% ) of e. coli isolates were positive for stx1 or stx2 genes , most of which occurred in spring ( p<0.05 ) . 
 there was no significant association between stec positivity and stool exam findings . 
 eighteen out of 27 ( 66.7% ) shiga toxin positive samples were isolated from males and the rest from females . 
 the most common stx - positive diarrheal samples showed loose consistency ( p<0.017).conclusionalthough the low frequency of stec in our population indicates that it is not a major problem in our population , stec should be regarded as an important infection because of its severe consequences . 
 further studies with greater sample size are needed to confirm our results . 
Introduction
Subjects and Methods
Bacterial isolation
DNA Extraction
Polymerase Chain Reaction
Amplicon detection
Statistical analysis
Findings
Discussion
Conclusion
Conflict of Interest