population studied - a total of 1,033 blood samples were drawn from 
 subjects from 51 rural communities in 12 of the 18 municipalities in the state of 
 quertaro . 2008 ) , housing conditions , co - habitation with 
 animals and seropositivity ( as reported by blood bank - based elisa testing ) ( cets 2002 ) . the committee of bioethics and research of the university of granada , spain approved 
 this study . blood sampling - blood samples were drawn during the epidemiological 
 interview from the cubital vein of either the right or left forearm after the area was 
 disinfected and cleaned using a cotton pellet soaked with a mixture of 5% glycerol in 
 ethanol . blood samples were collected in glass vacuum containers ( 7 ml ) containing gel 
 that inhibited blood clotting and prevented haemolysis . this precaution was important 
 because samples were frequently moved during transportation to the laboratory . blood was 
 centrifuged at 5,000 rpm for 10 min to separate serum from the globular fraction . serum 
 was stored in five 50-l aliquots and frozen at -20c until used . serological assays - determination of antibody expression in the 1,033 
 serum samples was performed using conventional assays . total extracts of the 
 epimastigote forms of t. cruzi ( biomrieux ) were used for 
 elisa testing and cut - off points were established for each series of sera studied ( lorca et al . relisa ( wiener lab ) 
 employed six antigens synthesised from trypomastigote and epimastigote fragments from 
 various t. cruzi strains ( sapa , 1 , 2 , 13 , 30 and 36 ) ( longhi et al . reactivity at dilutions > 
 1:8 was considered positive for iha ( wiener lab ) . the camargo method was 
 employed for ifa , using complete forms of t. cruzi epimastigotes 
 isolated from strain y ( of brazilian origin ) and strain t3 ( of mexican origin , state of 
 veracruz , derived from in vitro cultures in grace medium ) . samples with titres > 1:16 
 were considered positive ( camargo et al . 1986 , 
 berrizbeitia et al . 2012 ) . new diagnostic techniques - the iron superoxide dismutase ( fesod ) 
 method uses the isoenzyme fesod as an immunogenic protein for t. cruzi . this protein is extracted from the epimastigote and trypomastigote forms , which 
 were isolated from the colombian strain and cultured in vitro in grace medium 
 ( sigma ) supplemented with 10% bovine foetal serum ( v / v ) 
 ( gibco ) that had been previously heat inactivated at 56c for 30 min . flagellates were cultivated in 3 ml of medium in 25-cm plastic roux flasks 
 ( corning ) at 28c at an initial concentration of 5 x 10cells / ml . cultures 
 were centrifuged at 1,500 rpm for 10 min when the cellular concentration reached 
 approximately 2 x 10 cells / ml ( counted in a neubauer haemocytometer 
 chamber ) . the extracted enzyme was purified , fractioned and used as an antigen in 
 western blots to detect anti - t . cruzi 
 antibodies were detected at a dilution of 1:80 ( marn 
 & snchez - moreno 2010 , berrizbeitia et al . evaluations of statistically significant differences between the tests 
 were based on sensitivity , specificity , negative and positive predictive values , 
 concordance percentage and the kappa index . the following epidemiological variables were 
 considered in this study : presence of the vectors triatoma mexicana 
 ( 94% ) , triatoma dimidiate ( 3% ) , triatoma gerstaeckeri 
 ( 2% ) and triatoma pallidipennis ( 1% ) ( villagrn et al . 2009 ) , gender , age , housing type , co - habitation 
 with animals , trips outside the community and reported symptomatology that was 
 compatible with known signs and symptoms of the acute or chronic phases of chagas 
 disease . comparisons between epidemiologic parameters and survival patterns were 
 established in every studied population ( ferrer et al . statistical analyses were performed using spss v.11.5 to measure 
 confidence values and statistical significance between parameters . these data were collected using an epidemiological survey , which was completed at the 
 time of blood sample collection . the five immunological tests were performed in 1,033 samples and yielded the following 
 percentages of positive findings : elisa 8% , relisa 6.2% and iha and ifa 5.4% . the 8% 
 positive rate for fesod testing was consistent with the results of the most commonly 
 used antibody test ( fig . 1 , table i ) . 1 : immunological tests applied to samples of the studied individuals in 
 rural communities in the state of quertaro , mexico . fesod : iron superoxide 
 dismutase ; ifa : indirect immunofluorescence assay ; iha : indirect 
 haemagglutination assay ; relisa : recombinant elisa . table iseroreactive tests at trypanosoma cruzi in population of 
 different communities of the state of quertaro , mxicomunicipalitiesrural 
 communitiesseroreactive ( n ) 

 seropositivity at two 
 tests ( n)total persons sampled 
 ( n)elisarelisaihaifafesodsantiago de quertarorancho el pitayo11111131arroyo secotanchanaquito11001122el rejalgar110011pinal de amoleslas adjuntas20222260barranca del pltano211122el pltano111111landa de matamorosla aguita3111333peamilleralamitos32223387el piln423344cadereytaboyecito853378218llanitos743377tolimnpanales222222290san pablo422244horno de cal212222colnel gallo11911111111122viborillas110011el marqussan vicente ferrer954499109atongo223322tierra blanca210022huimilpanguadalupe 110001127las taponas111111santa teresa111111san juan del rosan juan del ro9610109949corregidoraalfaycan11111115joaqun herrera110011 

 total-8062545480801,033new diagnostic tool is considered , the iron superoxide dismutase ( fesod ) . ifa : indirect immunofluorescence assay ; iha : indirect haemagglutination 
 assay ; relisa : recombinant elisa . ifa : indirect immunofluorescence assay ; iha : indirect haemagglutination 
 assay ; relisa : recombinant elisa . all serological techniques are used primarily during the latent and chronic stages of 
 infection , when it is difficult to identify the parasites . cross - reactivity of chagas 
 disease tests with circulating antibodies related to other infections and diseases , such 
 as leishmaniasis , malaria , toxoplasmosis , hepatitis , leprosy , syphilis and collagen 
 vascular diseases , is common ( carvalho et al . this cross - reactivity inspired 
 us to conduct a study of five different serologic tests to determine the statistical 
 concordance among these tests and identify the true seroprevalence rate , as has been 
 previously described by the who ( foti et al . table iistatistical correlation between the five immunological tests used in the 
 study with the enzyme western blots - iron superoxide dismutase ( fesod ) match is 
 100%assayssensitivity ( % ) specificity ( % ) ppv ( % ) npv ( % ) concordance ( % ) kappa indexelisa r - elisa75.699.998.598980.8elisa - ifa68.299.998.397970.8elisa - iha63.510010097970.8elisa - fesod94.410098.4981000.96ifa : indirect immunofluorescence assay ; iha : indirect haemagglutination 
 assay ; npv : negative predictive values ; ppv : positive predictive values ; 
 relisa : recombinant elisa . ifa : indirect immunofluorescence assay ; iha : indirect haemagglutination 
 assay ; npv : negative predictive values ; ppv : positive predictive values ; 
 relisa : recombinant elisa . the statistical correlation rates between the five immunological tests were 8% between 
 elisa and fesod , 6.2% between elisa and relisa and 5.4% between iha and elisa - elisa - ifa , 
 showing that the sensitivity and specificity varied considerably . for example , recent recommendations 
 suggest the use of fresh sera and avoiding freezing and thawing samples that will be 
 used for relisa because sensitivity with respect to iha is lost . iha does not work with 
 antigenic extracts of t. cruzi strains that are obtained from humans ; 
 this test uses sensitised erythrocytes from sheep blood with antigenic extracts of the 
 epimastigote forms of cultured t. cruzi , which reduces sensitivity and 
 specificity . the predictive values and kappa index confirmed the usefulness of 
 performing two immunological tests for the detection of possible infections with this 
 haemoflagellate parasite . the survey also showed that infection is often present at a very young 
 age , which implies the establishment of the vector in the risk zone ( fig . fesod : iron superoxide dismutase ; ifa : indirect 
 immunofluorescence assay ; iha : indirect haemagglutination assay ; relisa : 
 recombinant elisa . a total of 8.8% of seroreactive inhabitants were of reproductive age and they performed 
 various activities . therefore , a socio - economic problem arises when these inhabitants 
 become infected because they are part of the economic support of their families . most of 
 the subjects in this study were women ( 74% ) and only 26% were men . the majority of individuals sampled in this study ( 83.3% ) were between the ages of 16 - 50 
 years old . this population often moves to other areas for work , study or leisure and 
 they are considered high risk . subjects between one-15 years of age made up 15.2% of the 
 sample and subjects between 51 - 95 years old constituted 1.5% of the sample ( fig . 3 : percentages of age groups in the studied population . 
  most of the population ( 99.3% ) did not travel outside their communities and remained in 
 the same locality for their entire lives . additionally , the majority of the population 
 ( 98% ) cohabited with animals and 8.7% had symptoms of infection , according to the survey 
 that was performed at the beginning of the study . the type of housing played an important role in the epidemiology of the infection 
 because building materials constitute an important variable in the establishment of the 
 vector . table iiipercentage of the types of material that forms part of ceilings , walls , 
 fences and floors wthat are part of the construction of the houses of 
 seropositive individuals studiedmaterialstraw ( % ) sheet ( % ) cardboard ( % ) wood ( % ) clay ( % ) adobe ( % ) ground ( % ) concrete ( % ) stone ( % ) roof70707020 - --10-wall88 - 15151010 - -90ground50 - ----506050 
  the only statistically significant difference noted in this study was in the percentage 
 of the different tests that were reactive , relative to symptomatology ( fig . fesod : iron superoxide dismutase ; ifa : indirect immunofluorescence assay ; iha : 
 indirect haemagglutination assay ; relisa : recombinant elisa . 
  the official mexican norm ( nom 1999 ) regarding 
 blood testing for chagas disease ( nom-033.ssa1-1997 ) mentions only that assays must be 
 performed in cities in geographical areas where chagas is endemic . the results obtained 
 between elisa - fesod was 8% , which indicates that infection by t. cruzi 
 in quertaro is five times higher than the national average of 1.6% ( guzmn - bracho 2001 ) . if this rate is extrapolated to the entire 
 population , an estimated 40,000 persons are infected , 30% of whom will develop cardiac 
 disease in the next 15 - 20 years . it is important to mention that quertaro should be 
 considered an endemic area because our results reflect a high percentage of high - risk 
 individuals and there is active transmission of infection ( 15.2% age paediatric ) . the 
 results have been reported to the health services of the state of quertaro . we were 
 also able to establish elisa as the test of choice for the detection of anti - t . cruzi antibodies for all persons who donate blood at the state centre of 
 haemotherapy . testing for t. cruzi should not be reserved for 
 individuals from endemic areas , despite possible controversy . similarly , the results obtained with elisa and fesod testing , which are sensitive and 
 specific for genus and species , found that the use of an enzyme - extracted parasite as 
 the antigen ( instead of antigenic extracts of epimastigote forms and/or trypomastigotes ) 
 cross - reacted with other aetiological agents ; this technique should therefore be avoided 
 because of the risk of false positive findings . new diagnostic techniques , such as the superoxide dismutase technique , have been 
 designed as confirmatory tests to overcome this problem . the superoxide dismutase assay 
 is the only diagnostic approach that can identify anti - t . . it should be 
 used in blood banks as the only definitive diagnostic test for the detection and 
 confirmation of reactive chagas infection and can eliminate the need to perform second 
 and third tests for the disease ( wendel & leiby 
 2007 , villagrn et al . the collected data show active transmission of t. cruzi in quertaro , 
 which was not previously considered an endemic zone .
immunological diagnostic methods for trypanosoma cruzi depend 
 specifically on the presence of antibodies and parasitological methods lack 
 sensitivity during the chronic and  indeterminate  stages of the disease . 
 this study 
 performed a serological survey of 1,033 subjects from 52 rural communities in 12 of 
 the 18 municipalities in the state of quertaro , mexico . 
 we detected anti - t . 
 cruzi antibodies using the following tests : indirect haemagglutination 
 assay ( iha ) , indirect immunofluorescence assay ( ifa ) , elisa and recombinant elisa 
 ( relisa ) . 
 we also performed western blot ( wb ) analysis using iron superoxide 
 dismutase ( fesod ) , a detoxifying enzyme excreted by the parasite , as the antigen . 
 positive test results were distributed as follows : elisa 8% , relisa 6.2% , ifa and iha 
 5.4% in both cases and fesod 8% . 
 a comparative study of the five tests was 
 undertaken . 
 sensitivity levels , specificity , positive and negative predictive values , 
 concordance percentage and kappa index were considered . living with animals , trips to 
 other communities , gender , age , type of housing and symptomatology at the time of the 
 survey 
 were statistically analysed using spss software v.11.5 . 
 detection of the fesod 
 enzyme that was secreted by the parasite and used as an antigenic fraction in wbs 
 showed a 100% correlation with traditional elisa tests .