
 study area and population - this study was performed in 2009 in the 
 communities of buriti seco and morro grande , which pertain to pedra preta , a little 
 village in a schistosomiasis endemic area of the rural region of montes claros , state of 
 minas gerais , brazil .
this area was chosen because the population shows a low migration 
 index ( fixed resident population ) and the infection rate in 2005 when the last treatment 
 occurred was 12% according to data provided by the control centre of diseases of montes 
 claros .
the populations of the two communities were included in this study , which 
 included 201 participants , 93 women and 108 men , aged between one-96 years . 

diagnostic techniques - an in depth analysis of stool samples , 
 including a combination of parasitological examinations , was performed before treatment 
 to establish the infection rate .
four stool samples from all participants were collected and analysed using the 
 kato - katz technique [ biomanguinhos - oswaldo cruz foundation ( fiocruz ) , 
 state of rio de janeiro , brazil ] and the commercial test tf - test 
 ( immunoassay com ind ltda , brazil ) as described by siqueira et al .
the pcr - elisa assay was used on 500 mg of the first 
 stool sample of each participant for comparison with the parasitological tests and 
 evaluation of diagnostic accuracy ( fig .
1:flowchart of stool examinations according to different diagnostic 
 techniques for establishing infection rate and the follow - up of positives after 
 treatment .
kato - katz 
  - the kk test was applied using 18 slides that were prepared as 
 follows : 12 slides of the first sample and two slides of the second , third and fourth 
 samples were analysed for a total of 750 mg of faeces for each participant ( 18 x 41.7 
 mg ) . 

tf - test 
  - the tf - test was performed on the first three samples 
 using portions of 500 mg , totalling 1,500 mg of faeces , which was processed with 3 ml of 
 ethyl acetate and one drop of detergent and centrifuged for 2 min at 500 
 g. 
 pcr - elisa assay - dna from 500-mg stool samples was isolated using the 
 commercial kit qiaamp dna stool mini kit , following the manufacturer s 
 recommendations ( qiagen gmbh , germany ) . a 121-bp repetitive sequence of s. 
 mansoni ( hamburger et al .
1991 ) was 
 amplified using a forward biotinylated primer ( 5-gatctgaatccgaccaaccg-3 ) and reverse 
 primer ( 5-atattaacgcccacgctctc-3 ) , according to the protocols described by gomes et al .
the amplicons were detected in 
 an elisa plate sensitised with streptavidin using a fluorescein 5-labeled probe 
 ( 5-tggtttcggagatacaacga-3 ) with available reagents in the commercial kit pcr plate 
 detection , according to the manufacturer s recommendations ( sigma , usa ) .
positive controls 
 extracted from s. mansoni eggs and negative controls ( water and 
 negative control of pcr ) were added to each pcr - elisa assay .
the receiver operating 
 characteristic curve ( roc ) determined the cut - off value of 0.116 .
the human beta - actin gene 
 was amplified using the primers aco1 ( 5-acctcatgaagatcctcacc-3 ) , biotinylated in the 
 5 region and aco2 ( 5-ccatctcttgctcgaagtcc-3 ) as a control for variations in the 
 efficiency of dna extraction and pcr - amplification .
products were detected using a 
 fluorescein 5-labeled probe ( 5-tctccttaatgcacgcacg-3 ) in the pcr - elisa system as 
 described by gomes et al .
( 2010 ) . one stool sample of each treated participant was analysed after treatment to establish 
 the cure rate using the kk technique examining 12 slides per sample and the pcr - elisa 
 assay , which used 500 mg of the same stool sample . 

comparisons of different diagnostic approaches - diagnostic parameters 
 were calculated using three different approaches for comparison ; ( i ) two - slide kk 
 examination , which is the current recommendation of the brazilian ministry of health for 
 the national schistosomiasis control program , ( ii ) 12-slide kk examinations to observe 
 the diagnostic parameters using the same amount of faecal material and ( iii ) the 
 reference test described above as an approximation of the real infection rate to 
 evaluate test performance . 
 statistical analysis - the software openepi , v.3.03 ( dean et al .
co - positivity and co - negativity values were calculated with 95% confidence intervals 
 ( ci ) for each of the three approaches .
the level of diagnostic agreement between 
 different diagnostic techniques was determined using the kappa coefficient .
landis and koch ( 1977 ) demonstrated poor concordance 
 when this coefficient was lower than 0.20 , weak concordance between 0.21 - 0.40 , moderate 
 concordance between 0.41 - 0.60 , good concordance between 0.61- 0.80 and excellent 
 concordance when higher than 0.81 . 

treatment of positives - all participants who were positive for 
 schistosomiasis as detected using the parasitological techniques were treated with a 
 single dose of 60 mg / kg praziquantel for children and 50 mg / kg for adults .
infections 
 with other helminths were treated using a single dose of 400 mg albendazole , as 
 proscribed by the brazilian ministry of health . 

cure assessment - cure assessments were performed 30 , 90 and 180 days 
 post - treatment .
sixty - nine of the 72 positive patients were followed up 30 days after 
 treatment , 67 patients were followed up after 90 days and 61 patients were followed up 
 after 180 days .
one stool sample from each participant was analysed using the kk 
 technique ( 12 slides per sample ) and the pcr - elisa assay , which was applied to 500 mg of 
 the same stool sample . 

ethics - the ethical research committee of the research centre ren 
 rachou / fiocruz ( cepsh / cpqrr 03/2008 ) and the national brazilian ethical board 
 ( scientific opinion 784/2008 , conep 14886 ) approved this study .
the objectives of this 
 study were presented and explained to all participants and written consent was obtained 
 from each individual , who signed a form prior to enrolment in this study .
seventy - two of the 201 stool samples examined were positive using the reference test . 

two - slide kk examination of the same sample , which is the current recommendation of the 
 brazilian ministry of health for the national schistosomiasis control program , detected 
 16 positives ( 8% ) , examination of 12 kk slides detected 29 positives ( 14.4% ) , 18 kk 
 slides detected 58 positives ( 28.9% ) , 32 positives ( 15.9% ) were detected using the 
 tf - test and 47 positives ( 23.4% ) were detected using pcr - elisa ( fig .
the comparison of pcr - elisa with two kk 
 slides , which corresponded to 83.4 mg of faeces , revealed that the co - positivity of 
 pcr - elisa was 93.75% ( ci :
71.67 - 98.89% ) and the co - negativity was 82.7% ( ci : 
 76.6 - 87.47 ) .
the comparison with 12 kk slides of the same sample , which corresponded to 
 500 mg of faeces ( 41.7 mg x 12 = 500 mg ) , demonstrated a co - positivity of pcr - elisa of 
 89.66% ( ci : 73.61 - 96.42% ) and a co - negativity of 87.8% ( ci : 82.06 - 91.87% ) ( table i )
2:diagnostic performance of parasitological and molecular techniques using 
 stool samples , pedra preta , municipality of montes claros , state of minas 
 gerais , brazil , 2009 .
table iresults of co - positivity and co - negativity for polymerase chain reaction 
 ( pcr)-elisa in relation to two kato - katz ( kk ) slides and in direct comparison 
 with 12 kk slides for the detection of infection with schistosoma 
 mansoni 
 kk 
 positivity rates % ( n / n)pcr - elisa % ( n / n ) 

 co - positivityco - negativity8 ( 16/201 ) ( 2 slides = 83.4 mg)93.7 ( 15/16)82.7 ( 153/185)14.4 ( 29/201 ) ( 12 slides = 500 mg)89.7 ( 26/29)87.8 ( 151/172 ) 
  out of 47 positives detected by pcr - elisa using 500 mg of the first sample , 12 were not 
 identified by the reference test .
thirty - seven positives only were detected by the 
 reference test , however the first assay was achieved with a single sample and the 
 parasitological combination with four samples .
two kk slides 
 showed moderate agreement with pcr - elisa ( kappa index of 0.405 ) and 12 slides showed a 
 substantial agreement ( kappa index of 0.616 ) ( table 
 ii ) .
the pcr - elisa assay showed moderate agreement with the reference test 
 ( kappa index of 0.426 ) ( table iii ) . 

table iicomparison and agreement between results detected by the polymerase chain 
 reaction ( pcr)-elisa in relation to kato - katz ( kk ) method using two and 12 
 slides of the same stool sample  kk ( 2 slides = 83.4 
 mg )  kk ( 12 slides = 500 
 mg )  positivenegativetotal  positivenegativetotalpcr - elisa ( 500 mg of the 1st 
 sample)positive153247positive262147negative1153154negative3151154 

 total16185201  29172201  kappa index : 0.405kappa index : 0.616 
 
 table iiicomparison and agreement between results detected by the reference 
 testa and polymerase chain reaction 
 ( pcr)-elisab 
 reference test 
 totalpositivenegativepcr - elisa ( 500 mg of the 1st 
 sample)positive351247negative37117154total72129201 
 kappa = 0.426 
 a : four samples = 18 slides of kato - katz + 
 tf - test ; b : single sample . 
 
 a : four samples = 18 slides of kato - katz + 
 tf - test ; b : single sample .
the cure rate 30 days after treatment using the kk technique was 100% and one positive 
 individual was detected using pcr - elisa ( cure rate of 98.5% ) .
three positive individuals 
 were detected using pcr - elisa 90 days after treatment ( cure rate of 95.5% ) , but these 
 patients were not identified using analysis of 12 kk slides .
the cure rate 180 days 
 after treatment was 98.4% using kk ( 1 out of 61 ) and 96.5% using pcr - elisa ( 2 out of 
 61 ) , with a final cure rate of 95.1% ( 3 out of 61 ) ( table iv ) . 

table ivassessment of cure 30 , 90 and 180 days after treatment by the kato - katz 
 ( kk ) and polymerase chain reaction ( pcr)-elisa techniquestechniquescure rate % (
n / n ) 

 30 days90 days180 dayskk ( 12 slides = 500 mg)100 ( 69/69)100 ( 67/67)98.4 ( 60/61)pcr - elisa ( 500 mg)98.5 ( 68/69)95.5 ( 64/67)96.5 ( 59/61 ) 

this assay proved a valuable 
 tool for the diagnosis of schistosomiasis despite its relatively high cost .
pcr - elisa 
 can be applied to comparisons between diagnostic methods and it is very useful to 
 confirm suspicious cases that were not detected using conventional parasitological 
 methods and evaluate cure control after chemotherapeutic treatment .
the pcr technique is widely used for the diagnosis of several human diseases , but its 
 application in neglected diseases , especially schistosomiasis , were explored only 
 recently .
2002 ) first described 
 the use of pcr in faecal samples for the diagnosis of schistosomiasis .
2009 ) compared to 
 routine parasitological examinations , such as the kk technique ( katz et al .
the positivity rate detected using pcr - elisa was 23.4% in the present study , which was 
 higher than the 14.4% using kk technique ( 12 slides ) .
however , three participants who 
 exhibited shedding eggs in faeces ( diagnosed using the kk technique ) were not identified 
 using pcr - elisa , which may be explained by the absence of eggs in the samples examined 
 by pcr assay , the presence of taq dna polymerase enzyme inhibitors or 
 failure in extraction , although the positive internal control ( human beta - actin gene ) 
 was detected in these samples .
further , the dna from these negative pcr - elisa samples 
 were diluted 1:10 , 1:20 and 1:40 and assayed again to exclude the possibility of 
 inhibiting the reaction , but the results were identical to the initial assay .
pcr - elisa detected 20 positive cases that were not identified using the kk technique ( 12 
 slides ) .
( 2009 ) who showed that pcr detected 23% positive cases amongst negative 
 samples for s. mansoni using the kk technique from individuals living 
 in a hypoendemic area .
these 
 authors reported that 16 out of 194 participants exhibited positive pcr , but did not 
 present eggs in faeces , whereas two positive participants using the kk method were 
 negative using pcr .
( 2003 ) 
 explained that the positive cases that were not detected using pcr could occur during 
 the dna amplification reaction as a result of various factors , such as amplification 
 inhibition by faecal compounds , dna degradation during transportation from the field to 
 the laboratory or the absence of eggs in the sample examined . a study by gomes et al .
( 2010 ) demonstrated that 
 pcr - elisa assay analysis of stool samples from 206 individuals from an endemic area 
 compared with 12 kk slides from a single sample revealed a sensitivity of 97.4% and 
 specificity of 85.1% , which are higher than the values in our study .
this difference may 
 be explained by factors that are inherent in the experimental procedures , which may 
 generate errors related to the detection of parasite dna , its degradation during 
 biological sample collection or absence in the sample analysed .
the need for 
 well - trained technicians who rigorously apply quality control measures at all stages of 
 the reaction should be stressed to avoid false - negative or false - positive results from 
 contamination .
molecular methods may suffer a loss of standardisation ( reproducibility ) and strength 
 when  in house 
these criteria are required for 
 the possible application of these methods in control programs ( gomes et al .
the commercial kit qiaamp dna stool mini kit was used in this 
 study for dna extraction from stool to avoid methodological interferences in the 
 procedure .
physical methods ( e.g. , separation of rooms and materials and the use of 
 laminar flow hood with ultraviolet light ) were utilised throughout the experiment to 
 minimise the chances of sample contamination .
therefore , it is indicated for 
 diagnosis of schistosoma spp in special situations , when high 
 sensitivity and specificity are required and infrastructure is available ( rabello et al .
a greater number of parasitic infections with low viral load are detected using 
 diagnostic methods with increased sensitivity .
seventy of the 72 ( 97.2% ) individuals 
 infected by s. mansoni presented low worm burden [ 1 - 100 eggs per gram 
 ( epg ) ] , one individual showed medium parasite burden ( 156 epg ) and another patient 
 showed a high intensity of infection ( 555 epg ) as evaluated using the reference 
 test .
the assessment of cure after treatment using the kk technique and pcr - elisa assay showed 
 a decrease of infected individuals .
for example , dna from dead worms or eggs remaining 
 in tissues can be excreted in faeces for a period of time after treatment .
another 
 reason is that the sensitivity of the pcr - elisa for the detection of low parasite 
 burdens is greater than the kk technique ( wichmann et 
 al .
the lower cure rate six months after treatment may be explained by 
 the temporary interruption of the oviposition by female worms as a result of treatment 
 and a resumption of oviposition at a later time .
these individuals also may have been 
 re - infected because of the constant exposure they may suffer in the endemic areas .
managers of control programs in areas of low transmission must choose between the kk 
 technique with various samples or pcr - elisa assay to detect the largest number of 
 infected individuals using a single sample .
notably , the first technique is less 
 expensive , but the latter technique is more accurate .
the cost of pcr reagents is us $ 10 
 per faecal sample ( gomes et al . 2010 ) , but these 
 costs are greatly reduced when the technique is utilised on a large scale .
pcr also 
 provides good precision ( reproducibility and repeatability ) , is genus - specific and may 
 estimate worm burden , which correlates with the values of epg of faeces determined using 
 the kk technique .
the results obtained using pcr - elisa support the development of a promising diagnostic 
 method that only uses a faecal sample .
this technique represents an important step 
 forward to support studies on epidemiology and disease control , especially when the cost 
 would be reduced by the use of industrial scale production .
the assessment of cure in 
 situations of very low parasite load , such as after therapeutic interventions , may 
 require increased numbers of kk slides or a test with higher sensitivity than pcr - elisa . 

this study also demonstrated a low positivity rate during follow - up after treatment , 
 which indicates that an in - depth diagnostic survey is more effective and it may likely 
 prevent successive treatments .
 this study evaluated parasitological and molecular techniques for the diagnosis and 
 assessment of cure of schistosomiasis mansoni . 
 a population - based study was performed 
 in 201 inhabitants from a low transmission locality named pedra preta , municipality 
 of montes claros , state of minas gerais , brazil . 
 four stool samples were analysed 
 using two techniques , the kato - katz ( kk ) technique ( 18 slides ) and the 
 tf - test , to establish the infection rate . 
 the positivity rate of 18 kk 
 slides of four stool samples was 28.9% ( 58/201 ) and the combined parasitological 
 techniques ( kk+tf - test ) produced a 35.8% positivity rate ( 72/201 ) . 
 
 furthermore , a polymerase chain reaction ( pcr)-elisa assay produced a positivity rate 
 of 23.4% ( 47/201 ) using the first sample . 
 all 72 patients with positive 
 parasitological exams were treated with a single dose of praziquantel and 
 these patients were followed - up 30 , 90 and 180 days after treatment to establish the 
 cure rate . 
 cure rates obtained by the analysis of 12 kk slides were 100% , 100% and 
 98.4% at 30 , 90 and 180 days after treatment , respectively . 
 pcr - elisa revealed cure 
 rates of 98.5% , 95.5% and 96.5% , respectively . 
 the diagnostic and assessment of cure 
 for schistosomiasis may require an increased number of kk slides or a test with 
 higher sensitivity , such as pcr - elisa , in situations of very low parasite load , such 
 as after therapeutic interventions . 
SUBJECTS, MATERIALS AND METHODS
RESULTS
DISCUSSION