we studied 2 outbreaks of orally transmitted chagas disease ( 120 cases , 5 deaths ) . the first occurred in chichiriviche , vargas state , a coastal community ( population 800 persons ) 50 km northwest of caracas , venezuela . the outbreak occurred at a primary school where food was prepared on site . in early april 2009 , a total of 71 children ( 613 years of age ) who attended the morning school shift and 14 adults became ill . exposure of these persons to t. cruzi was established by use of igm and igg elisa . parasitemia was observed in 33 of the patients with serologic results positive for t. cruzi infection ( 9,10 ) . the second outbreak occurred in antimano , a peri - urban slum southwest of central caracas ( technical appendix figure 1 ) . in may 2010 , 35 patients with suspected t. cruzi infection were examined at hospital miguel perez carreno in caracas . patients reported that they regularly ate at the same local communal canteen . among the patients tested , 15 were positive for t. cruzi igm and igg ( 9 ) . parasitemia in 14 patients was confirmed indirectly by hemoculture . of the 35 patients , 21 ( 2 adults , 19 children ) , we undertook intensive additional sampling of contemporary , nonhuman sources local to each outbreak and of human and nonhuman sources from more distant localities throughout venezuela . in total , 246 t. cruzi strains and clones were typed for 23 microsatellite markers ( technical appendix 1 table ) ( 8) . a list of the samples and their sites of origin is in technical appendix 2 ) . individual level sample clustering was defined first by constructing a neighbor joining tree based on pairwise distances between multilocus genotypes ( figure 1 ) . a second analysis used k - means clustering and discriminant analysis of principal components ( figure 2 ) ( 11 ) . to assess connectivity between human and nonhuman outbreak cases , population - level genetic diversity was assessed first by calculating allelic richness then private allele frequency over loci between each human  nonhuman population pair ( table 2 ) . unrooted neighbor joining tree showing genetic clustering among trypanosoma cruzi isolates from 2 outbreaks of oral disease in northern venezuela . based on pairwise genetic distances ( 1  proportion of shared alleles ) between multilocus microsatellite profiles ( 23 loci ) generated from 246 isolates and clones . indicate isolates associated with the outbreaks . discriminant analysis of principal components showing genetic clustering among trypanosoma cruzi isolates from 2 outbreaks of oral disease in northern venezuela . ellipses correspond to the optimal ( as defined by the bayesian information criterion minimum ) number of population clusters among the genotypes analyzed . images indicate sample host origin ( human , rodent , marsupial , or triatomine ) , while colors correspond to the key in figure 1 . a full list of samples and population assignments ( numbered boxes ) is included in technical appendix 2 . lower left shows linearized fst ( genetic differentiation ) values ; upper right shows associated p values . clustering results determined by discriminant analysis of principal components and neighbor joining were broadly congruent . in the former , substantial overlap existed between some clusters , especially those from capital and vargas states ( e.g. , those labeled x  in figure 2 ) , while others were highly distinct ( e.g. , cluster 8 in figure 2 ) . human isolates from both oral outbreaks are extremely distinct from non - orally transmitted isolates collected from humans throughout venezuela . almost all these presumably vector - transmitted strains are closely related to one another , despite their geographic dispersal ( cluster 8 in figure 2 ) . by comparison , oral outbreak strains that were isolated a mere 50 km apart ( clusters 2 , 5 , 7 , 9 , 1517 ) are far more globally diverse . unlike most human isolates in venezuela , which are distinct from nonhuman strains , oral outbreak isolates are interspersed among nonhuman strains from venezuela . furthermore , samples from both outbreaks clustered among nonhuman strains local to that outbreak , clearly indicating a local origin . oral samples from each outbreak are polyphyletic with respect to strains from their immediate environment , a finding consistent with multiple contamination events or multiclonal infection sources . fst values further support connectivity between outbreak and local environmental samples in both antimano and chichiriviche ( table 1 ) . a lack of private alleles between human and nonhuman isolates also supports a local source for the chichiriviche outbreak ( table 2 ) . fst values in the 4-way comparison between outbreak and local environmental strains are , however , somewhat equivocal with respect to the entire dataset ( table 1 ) . cluster analysis showed that the human and nonhuman strains from chichiriviche interspersed with strains from other states in venezuela ( figure 2 ) . thus , we can not confirm a uniquely local origin for the chichiriviche outbreak , despite a low value for fst , and it is possible that some contaminating strains originated elsewhere . this study demonstrates the value of rigorous molecular epidemiologic analysis of orally transmitted t. cruzi outbreaks , including the importance of appropriate sampling to identify the origin of the infecting strains . the foodstuff that propagated the peri - urban outbreak in antimano was certainly contaminated locally . an active nonhuman transmission cycle in the slums of caracas , maintained by rattus rattus rodents and panstrongylus geniculatus triatomines , is the likely source . the chichiriviche outbreak , however , has potential sources both in and outside the immediate area . as found in chagas disease outbreaks linked to aa palm fruit in brazil ( 12 ) , the t. cruzi parasite can survive for several days in some foodstuffs ( 13 ) . also , triatomines can survive for months in harvested crops ; thus , multiple hygiene interventions are potentially necessary along the food production line ( 14 ) . nonetheless , if the foodstuff implicated was prepared locally , local contamination represents the most likely source of the outbreak . crucial to understanding parasite transmission in general , we believe , are genetic differences between strains from orally and non - orally transmitted human cases . all tci strains appear to be infective to humans and adapted to long - term carriage ( 8) . however , the presence of a common , reduced - diversity tci genotype cluster ( tcidom ) among a high proportion of human chagas disease cases in south america is also well established ( 7,8 ) . we originally hypothesized that tcidom was maintained , despite the presence of sympatric and infective sylvatic strains , because of low parasite transmission efficiency by invasive sylvatic vectors ( 8) . thus , unlike tcidom strains , those from orally transmitted t. cruzi cases demonstrated high genetic diversity and clearly originated from local nonhuman t. cruzi populations . however , it is also true that all tcidom strains we isolated originated from patients with chronic infection , and all orally transmitted cases were in the acute phase . we can not , therefore , rule out a role for immune selection in driving the frequency of tcidom infections among humans ; such selection represents an intriguing topic for future enquiry . molecular tools and reference datasets are now available to determine the source of acute chagas disease outbreaks within days of their occurrence . the plummeting cost of such analyses means it is time to apply population genetic techniques and markers developed for trypanosomes as genuine epidemiologic tools . photo of antimano , a suburban slum of caracas , map showing the geographic distribution of trypanosoma cruzi i isolates analyzed , and primers and chromosomal positions for microsatellite loci used in a study of the molecular epidemiologic source tracking of 2 outbreaks of oral chagas disease , venezuela . trypanosoma cruzi isolates and clones analyzed in a study of the molecular epidemiologic source tracking of 2 outbreaks of oral chagas disease , venezuela .