
 location of the study area - 
 the administrative district of barcelos is located north of am . to the east , it borders the state of roraima ; to the southeast and south , it borders the district of santa isabel do rio negro and , to the north , it borders venezuela ( latitude 058'1 " south of the equator and longitude 6256 " west of greenwich ) .
the city of barcelos , where this study was conducted , is located on the right bank of the negro river , 490 km from manaus , the capital of am ( 
 fig . 2 
 ) . 

2location of barcelos , state of amazonas , brazil , 2010 , and number of blood samples collected by teams ( a - f ) and city district for screen tests . 
 

survey and samples - 
 the new serological survey was conducted in the main settlement of barcelos and covered the entire resident population that was willing to participate in the survey after providing informed consent . for minors ,
the main settlement of the municipality was divided into six areas ( a - f ) , which were to be covered by the municipality 's health agents under supervision by two laboratory technicians at the municipal hospital .
the teams were trained by the project coordinator ( jr coura , a physician and infectologist ) and were monitored throughout the survey by a postgraduate student ( mhp marquez ) .
each team was composed of two health agents ; the two laboratory technicians each supervised three of the teams and all six teams were monitored by the postgraduate student . in total , 4,880 blood samples were collected on filter paper in accordance with the technique of souza and camargo ( 
 1966 
 ) for the screening test : 770 samples were collected by team a ( so sebastio ) , 925 by team b ( so lzaro ) , 1,000 by team c ( so francisco ) , 728 by team d ( nazar ) , 817 by team e ( vila linhares ) and 640 by team f ( aparecida ) . all the blood samples on the filter paper were eluted in 250 l of phosphate - buffered saline ( ph 7.2 ) and were tested using the iif test , as recommended by fife and muschel ( 
 1959 
 ) and souza and camargo ( 
 1966 
 ) , as adapted by ostermayer et al .
( 
 2011 
 ) for the detection of anti- 
 t. cruzi 
 antibodies .
the iif - chagas kit produced by biomanguinhos was used . in this kit , the antigens used are cultured epimastigote forms of 
 t. cruzi 
 in liver infusion tryptose medium .
the tests were performed in the serology sector of the parasitic diseases laboratory of the oswaldo cruz institute , oswaldo cruz foundation , state of rio de janeiro ( rj ) , brazil .
the slides were prepared in accordance with the classical techniques for iif and were examined under an immunofluorescence microscope by at least two observers .
the slide readings were considered to be negative ( unreactive , absence of fluorescence ) , doubtful ( weakly reactive , low intensity of fluorescence ) or positive ( strongly reactive , high intensity of fluorescence ) . 

confirmatory serological test - 
 to perform confirmatory reactions , venous blood ( 5 ml ) was collected by the laboratory technicians from 315 patients ( 60% ) who presented positive ( 137 patients ; 62% ) or doubtful ( 178 patients ; 59% ) iif reactions to 
 t. cruzi 
 infection based on the serological screening of blood on the filter paper .
after separation , the serum was frozen , packed on dry ice and sent from barcelos to our laboratory in rj .
these serum samples were subjected to three serological reactions : iif ( chagas kit , biomanguinhos ) with a dilution of 1:40 , with the positive samples tittered as much as 1:1,280 , conventional elisa ( dmed kit ) and recombinant elisa ( wiener lab .
samples were considered to be seronegative if two or three of the tests were negative and seropositive if two or three of the tests were positive .

 serological screening test - 
 the screening test performed on the blood placed on the filter paper using immunofluorescence , 221 ( 4.5% ) of the 4,880 samples were considered to be positive ( strongly reactive ) and 302 ( 6.2% ) were considered to be doubtful ( weakly positive ) ( 
 table ii 
 ) .
of the 4,880 samples examined , 2,741 ( 56.2% ) were from females and 2,139 ( 43.8% ) were from males . among the 221 samples that were considered to be positive , 104 ( 3.8% ) were from females and 117 ( 5.5% ) were from males .
the numbers of blood samples collected on filter paper , along with the percentage of positivity for 
 t. cruzi 
 infection , varied according to the number of people living in the area and their degree of exposure to infection in rural areas , especially in relation to piassava harvesters and their families .
this characteristic has already been presented in previous studies by our group ( 
 coura et al .
1995a 
 , 
 b 
 , 
 2002a 
 , 
 b 
 , 
 1999 
 , 
 albajar et al .
2010 
 , 
 coura & junqueira 2012 
 , 
 junqueira et al . 2013 
 ) . 
 table ii   age groups 
 ( years )   tested 
 n positives 
 n ( % ) doubtful 
 n ( % ) < 5 495 9 ( 1.8 ) 17 ( 3.4)5 - 962618 ( 1.9)30 ( 4.8)10 - 1462212 ( 2.9)30 ( 4.8)15 - 1952424 ( 4.6)35 ( 6.7)20 - 2987246 ( 5.3)56 ( 6.4)30 - 3957834 ( 5.9)41 ( 7.1)40 - 4941324 ( 5.8)39 ( 9.4)50 - 5929319 ( 6.5)18 ( 6.1)60 - 6922717 ( 7.5)17 ( 7.5)70 - 791389(6.5)10 ( 7.2 ) > 79554 ( 7.3)2 ( 3.6)unknown 
 37 5 ( 13.5)7 ( 18.9 ) 4,880   221 ( 4.5 )   302 ( 6.2 ) 
 a 
 : yanomami indians . 
 
 a 
 : yanomami indians .
the prevalence of serological positivity in samples collected on filter paper gradually increased after the age of 15 years , precisely when adolescents ( especially males ) started to be involved in piassava harvesting and after the age of 20 years among women , when as wives would accompany their husbands to the piassava plantations ( 
 fig .
the unknown age group ( yanomami indians who do not know their age ) is the most exposed to wild triatomines in the forest ; therefore , they had the highest percentage of iif - positive results ( 13.5% ) , as shown in 
 fig .
3percentage of positive immunefluorescence by age group in the screen tests in barcelos , state of amazonas , brazil , 2010 . 
 

confirmatory serological test - 
 among the 137 serum samples that were considered to be positive based on immunofluorescence testing , 37 ( 27% ) were considered to be seronegative based on the confirmatory serological test , 67 ( 48.9% ) were considered to be serodivergent , as only one of the three reactions was positive , and only 33 ( 24.1% ) were confirmed as seropositive , with two or three positive reactions ( 
 table iii 
 ) .
of the 178 serum samples for which the immunofluorescence was considered doubtful , 89 ( 50% ) were seronegative in all three confirmatory reactions , 79 ( 44.4% ) were considered to be serodivergent 
 , 
 with only one positive reaction out of the three reactions , and only 10 ( 5.6% ) were considered to be seropositive , with two or three positive reactions ( 
 table iii 
 ) . in summary , of the 137 samples that were positive in the screening serological tests , only 33 ( 24.1% ) were positive in the confirmatory tests , while only 10 ( 5.6% ) of the 178 samples that were doubtful in the screening test were positive in the confirmatory test , as shown in 
 table iii 
 .
the numbers of serum samples and the titres of igg confirmed by immunofluorescence as positive are shown in 
 fig . 4 
 .
these data most likely reflect the low level of parasitaemia and the particular 
 t. cruzi 
 strain circulating in the area ( tci ) , which likely provide low stimulation for antibody genesis . 

table iiiconfirmatory serological tests of 137 samples positives and 178 doubtful by indirect immunofluorescence ( iif ) in blood from filter paper evaluated by iif and elisa conventional and or recombinantserological evaluation   positives iif 
 from filter paper 
 n ( % ) doubtful iif 
 from filter paper 
 n ( % ) seronegatives 37 ( 27 ) 89 ( 50)serodivergents 67 ( 48.9)79 ( 44.4)seropositives33 ( 24.1)10 ( 5.6 ) total   137 ( 100 )   178 ( 100 ) 
 
 fig .
4titres of igg of immunofluorescence in serum from confirmatory serological tests in barcelos , state of amazonas , brazil , 2011 .
the high prevalence found in the preliminary serological screening surveys that we conducted in 1991 , 1993 and 1997 ( 
 coura et al .
1999 
 ) certainly occurred due to cross - reactions with a variety of diseases that are endemic in this area , including malaria , american cutaneous leishmaniasis , tuberculosis , leprosy and 
 trypanosoma rangeli 
 infection .
additionally , the low titres of anti- 
 t. cruzi 
 antibodies demonstrated in the present study favoured occurrences of cross - reactions with other infections .
the prevalence of 
 t. cruzi 
 infection in barcelos was observed to vary according to the intensity of exposure . in a group of 244 highly exposed individuals
, we found that 11% were serologically positive ( iif and tesa - blot ) .
however , among the 46 individuals in this group who underwent xenodiagnosis and polymerase chain reaction ( pcr ) analysis , we only isolated 
 t. cruzi 
 in 19% of the individuals through xenodiagnosis and the pcr was only positive in these cases ( coura et al .
( 1998a , b ) demonstrated only two lineages of 
 t. cruzi 
 in the area ( tci and z3 ) .
although no studies have provided evidence , we can suppose that these lineages of 
 t. cruzi 
 are poorly immunogenic for antibody generation .
finally , we recommend that the serological diagnosis of 
 t. cruzi 
 infection in the amazon region should be made using at least two different techniques , e.g. , immunofluorescence and elisa and confirmed by western blot analysis when possible .
 the serology of human 
 trypanosoma cruzi 
 infection in the rio negro microregion is very complex because of the large numbers of false - positive cases that result from low antibody titres and cross - reactions with other infections . in the present study , 
 we collected 4,880 blood samples on filter paper ; of these , indirect immunofluorescence ( iif ) was strongly reactive in 221 ( 4.5% ) , which were considered to be positive ( iif strongly reactive ; high intensity of fluorescence ) and weakly reactive in 302 ( 6.2% ) , which were considered to be doubtful ( iif weakly reactive ; low intensity of fluorescence ) . 
 the confirmatory test on the serum using at least two of three techniques ( iif , conventional elisa and recombinant elisa ) on 137 samples that were positive in the screening test only confirmed 33 cases ( 24.1% ) . 
 of the 178 samples that were considered doubtful in the screening test , only 10 ( 5.6% ) were considered to be positive in the confirmatory test . 
 finally , we recommend that the serological diagnosis of 
 t. cruzi 
 infection in the amazon region be made using at least two different techniques , for example immunofluorescence and elisa and confirmed by western blot analysis when possible . 