research article comparative study of the accuracy of...

Research ArticleComparative Study of the Accuracy of Different Techniques forthe Laboratory Diagnosis of Schistosomiasis Mansoni in Areas ofLow Endemicity in Barra Mansa City Rio de Janeiro State Brazil

Maria Cristina Carvalho Espiacuterito-Santo12 Moacutenica Viviana Alvarado-Mora3

Pedro Luiz Silva Pinto4 Maria Carmen Arroyo Sanchez5 Emmanuel Dias-Neto67

Vera Luacutecia Pagliusi Castilho8 Elenice Messias do Nascimento Gonccedilalves8

Pedro Paulo Chieffi910 Expedito Joseacute de Albuquerque Luna10 Joatildeo Renato Rebello Pinho3

Flair Joseacute Carrilho1 and Ronaldo Cesar Borges Gryschek1

1 Department of Infectious and Parasitic Diseases and Laboratory of Immunopathology of Schistosomiasis (LIM-06)School of Medicine University of Sao Paulo 05403-000 Sao Paulo SP Brazil

2 University Center of Volta Redonda 27240-560 Volta Redonda RJ Brazil3 Department of Gastroenterology and Laboratory of Tropical Gastroenterology and Hepatology School of MedicineUniversity of Sao Paulo 05403-000 Sao Paulo SP Brazil

4 Department of Enteroparasites at the Parasitology and Mycology Service from the Adolfo Lutz Institute01246-902 Sao Paulo SP Brazil

5 Laboratory of Seroepidemiology and Immunobiology Institute of Tropical Medicine University of Sao Paulo05403-000 Sao Paulo SP Brazil

6 Laboratory of Medical Genomics AC Camargo Cancer Center 01509ndash010 Sao Paulo SP Brazil7 Institute of Psychiatry (LIM-27) Sao Paulo Medical School University of Sao Paulo 01246-903 Sao Paulo SP Brazil8 Section of Parasitology Central Laboratory Division of Hospital das Clınicas School of Medicine University of Sao Paulo05403-000 Sao Paulo SP Brazil

9 Santa Casa Medical School 01221-020 Sao Paulo SP Brazil10Institute of Tropical Medicine University of Sao Paulo 05403-000 Sao Paulo SP Brazil

Correspondence should be addressed to Maria Cristina Carvalho Espırito-Santo cristinasantouspbr

Received 2 March 2015 Revised 6 May 2015 Accepted 10 May 2015

Academic Editor Stephan Karl

Copyright copy 2015 Maria Cristina Carvalho Espırito-Santo et al This is an open access article distributed under the CreativeCommons Attribution License which permits unrestricted use distribution and reproduction in any medium provided theoriginal work is properly cited

Schistosomiasis constitutes a major public health problem with an estimated 200 million people infected worldwide Manyareas of Brazil show low endemicity of schistosomiasis and the current standard parasitological techniques are not sufficientlysensitive to detect the low-level helminth infections common in areas of low endemicity (ALEs) This study compared theKato-Katz (KK) Hoffman Pons and Janer (HH) enzyme-linked immunosorbent assay- (ELISA-) IgG and ELISA-IgM indirectimmunofluorescence technique (IFT-IgM) and qPCR techniques for schistosomiasis detection in serum and fecal samples usingthe circumoval precipitin test (COPT) as reference An epidemiological survey was conducted in a randomized sample of residentsfromfive neighborhoods of BarraMansa RJ with 610 fecal and 612 serum samples ELISA-IgM (214) showed the highest positivityand HH and KK techniques were the least sensitive (08) All techniques except qPCR-serum showed high accuracy (82ndash955)differed significantly from COPT in positivity (119875 lt 005) and showed poor agreement with COPT Medium agreement was seenwith ELISA-IgG (Kappa = 0377) and IFA (Kappa = 0347) Parasitological techniques showed much lower positivity rates thanthose by other techniques We suggest the possibility of using a combination of laboratory tools for the diagnosis of schistosomiasisin ALEs

Hindawi Publishing CorporationBioMed Research InternationalVolume 2015 Article ID 135689 16 pageshttpdxdoiorg1011552015135689

2 BioMed Research International

1 Introduction

Schistosomiasis is a major public health problem with 200million people infected worldwide and 700 million peopleresiding in areas of infection risk [1 2]

In Brazil schistosomiasis has been reported to occur in19 states and it is estimated that approximately 6 millionpeople are infected and 25 million are at risk of contractingthe diseaseThenational positivity rate is 694 ranging from004 in Piauı State to 1188 in Pernambuco State In Rio deJaneiro State the positivity rate is 156 [3]

Brazil has areas of different prevalence rates varying fromstate to state as shown in Figure 1 [3]

Of the various known species of Schistosoma S mansonihas the widest global distribution and is the only species thatcauses schistosomiasis in Brazil [4]

Although the serious forms of schistosomiasis havebecome less prevalent thanks mainly to the implementationof mass chemotherapy the geographic expansion of schisto-somiasis continues apace with the expansion of agriculturalzones and irrigated areas [5]

The classification of the individual infection intensitycriteria for S mansoni is estimated by the quantity of eggsobserved in parasitological examination of feces using theKato-Katz (KK) technique [2] According to WHO 2002 [6]the infection classification is high parasitic load (ge400 epg)medium parasite load (100ndash399 epg) and low parasite load(lt100 epg) and areas with high medium or low endemicityshow prevalence of ge50 ge10 lt50 or lt10 respectivelyIn ALEs approximately 75 of infected individuals areasymptomatic show few symptoms and have low parasiteload which hinders diagnosis [6]

The state of Rio de Janeiro presents the lowest numberof confirmed cases and deaths due to schistosomiasis in thesoutheast region of Brazil [3 7] The city of Barra Mansa isdefined as microregion 2 of the Vale do Medio Paraıba Riode Janeiro State [8] The city of Barra Mansa is located in thesouthern part of the state of Rio de Janeiro

Barra Mansa is one of the foci of S mansoni infectionin the state of Rio de Janeiro [8] The average prevalencewas estimated to be 1 from 2001 to 2008 based on thecases reported by the Notifiable Diseases Information System(SINAN) from 2001 to 2008 [9]

The endemic foci lie within the urban perimeter Theneighborhood of Siderlandia shows the highest prevalencefollowed by the neighborhoods of Santa Clara Sao LuizCantagalo andNova Esperanca Isolated cases of infection byS mansoni have been reported in further 30 neighborhoods[9]

Detection of S mansoni eggs in feces has historicallybeen used as the reference for diagnosing schistosomiasisand Schistosoma species are identified by their characteristicmorphology showing a lateral spicule The parasitologicalmethods are highly specific inexpensive and relatively sim-ple to execute [2 10ndash12] The Kato-Katz (KK) techniqueis most commonly used for detecting S mansoni eggs inepidemiological studies allowing the quantification of eggsin fecal samples The Hoffman technique (HH) is basedon spontaneous sedimentation and it is effective because

0 250 500 1000

(km)

W E

N

S

No data

lt5

gt15

Positivity ranges ()2003ndash2012

5ndash15

Figure 1 Distribution of positivity ranges for schistosomiasis basedon the record of cases on investigated cities Brazil 2012 SourceSISPCE-SVSMS

embryonated S mansoni eggs are heavy however it is notsuitable for quantification of eggs in feces

Although these parasitological methods are inexpensiveand simple to perform they lack sensitivity especially inALEs [13ndash18] The Secretariat of Health Vigilance in Brazilhas proposed the elimination of this form of helminthiasisTherefore there is a need to define alternative laboratorydiagnostic techniques for detection of S mansoni in ALEsThus the aim of this study was to compare the efficiencyof existing parasitological immunological and moleculardiagnostic methods in areas of low prevalence of S mansoniusing the circumoval precipitin test (COPT) as a referencedue to its high sensitivity and specificity in ALEs [19 20]

2 Materials and Methods

21 Study Design Population and Sample Size S mansoniis endemic in the city of Barra Mansa Rio de Janeiro StateBrazil with an estimated prevalence of 1 [9] Data for2001ndash2008 from the Notifiable Diseases Information System(SINAN) showed that the disease is most prevalent in theneighborhoods of Siderlandia Santa Clara Sao Luiz Nova

BioMed Research International 3

Esperanca and Cantagalo which belong to the Barra MansaRiver Basin a tributary of the Paraıba do Sul RiverThese fiveneighborhoods located on the outskirts of the city of BarraMansawere selected for this cross-sectional study Samples offeces and serum were collected from April to December 2011

The sample size was calculated assuming a prevalence of1 with an addition of 30 to compensate for losses Theestimated sample size required was 650 individuals residingin the above neighborhoods Households were systematicallyselected (one in six) and individuals were randomly selectedby a draw among those who agreed to participate in the studySubjects who were older than 5 years of age and had notbeen treated for S mansoni in the last year were eligible forinclusion

22 Statistical Analysis Statistical analysis was performedusing SPSS (Statistical Package for the Social Sciences) forWindows version 150 (SPSS Inc Chicago IL USA) andMicrosoftExcel 2003 Significance levels were fixed by accept-ing a Type 1 error of 5 (120572 = 005) Population characteristicswere described using absolute and relative frequencies andcalculation of mean ages and standard deviations All partic-ipants were evaluated by each diagnostic technique

Each S mansoni infection measurement technique wascompared and marginal associations were verified usingMcNemarrsquos test Pairwise concordance between results wasassessed using Cohenrsquos kappa index and 95 confidenceintervals The Kappa index values range interpretation wasas follows poor agreement (lt020) low agreement (020 to040) moderate agreement (041 to 060) good agreement(061 to 080) and very good agreement (081 to 100) [21]

Associations between S mansoni infection and age rangesex neighborhood river water use and history of schistoso-miasis were assessed for each technique using the Chi-squaretest Fisherrsquos exact or likelihood ratio tests

We compared the accuracy (sensitivity specificity likeli-hood ratio and predictive values) of serological techniques tothose of parasitological techniquesWe also compared resultsamong techniques to determine which were most effective indiagnosing S mansoni in areas with a similar epidemiologicalprofile to the target area of this study

23 Ethical Aspects In accordance with the rules governinghuman subject research informed consent was obtainedto meet the recommendations of Resolution n∘ 466 fromDecember 12 2012 of the National Council of HealthThis research project was approved by the Research EthicsCommittee of the Department of Infectious and ParasiticDiseases of the Faculty of Medicine of the University of SaoPaulo and the Research Ethics Committee of the Hospitaldas Clınicas (CAPPesq) of the Faculty of Medicine of theUniversity of Sao Paulo (Approval number 040509)

The experimental research proceduresmet Laws 663879and 960598 Decree 2464534 the Ethical Principles ofAnimal Experimentation the Principles for Research Involv-ing Animals [22] and other directives governing animalresearch The study began after approval of research projectnumber CEP-IMT 2011096 by the Animal Ethics Research

Committee of the Institute of Tropical Medicine of Sao PauloUniversity of Sao Paulo Brazil

24 Methods of Diagnostic Investigation This was an inter-disciplinary study and the research laboratories developedtheir activities independently following the conditions andoperationalization timelines that respected the routine ofeach institute involved Samples from all individuals in thestudy were subjected to the diagnostic techniques describedand the reference diagnostic technique (COPT) [23] Theprofessionals who standardized and interpreted the COPTdid not know the results for the diagnostic techniques thusreducing any interpretation bias

The participating laboratories and the diagnostic tech-niques they developedwere as followsMunicipal Secretary ofHealth of BarraMansaRJ (feces and serum collections) Cen-troLab Laboratory Volta RedondaRJ (preparing sample pre-analysis) Institute Adolfo Lutz (COPT and ITF-IgM) Insti-tute of Tropical MedicineUSP (ELISA) Laboratory of Gas-troenterology and Tropical Hepatology at the Departmentof GastroenterologyFMUSP (molecular biology) ResearchCenter at AC Camargo Hospital (molecular biology) ReneRachou Research CenterFIOCRUZ Belo HorizonteMG(molecular biology) Parasitological Section of the CentralLaboratory Division HCFMUSP (KK and HH)

The standardization and application of the diagnostictechniques were developed from December 2010 to Decem-ber 2012 when the statistical analyses began

25 Methods for Laboratory Diagnosis The Family HealthProgram and health agents of the Municipal SchistosomiasisControl Program (PCE) collected 610 randomized fecalsamples and 612 serum samples Of these 572 sampleswere paired from inhabitants of 5 peripheral neighborhoodsof Barra Mansa Rio de Janeiro The serum samples werealiquoted and stored at minus20∘C and then transported to SaoPaulo in thermal boxes containing dry ice and stored atminus20∘Cin the lab until further testing

26 Parasitological Methods Stool samples were preparedfollowing the KK (Helm Test Bio-Manguinhos Fiocruz Riode Janeiro RJ Brazil) and HH techniques and stored at4∘C until shipment For the KK technique two slides wereprepared and stored in boxes fixed with a rigid polypropylenecover lined with cork (Prolab-Prolab Sao Paulo SP Brazil)For the HH technique samples were preserved in 10formalin (Indalabor Indaia Laboratorio Farmaceutico LtdaDores do Indaia Minas Gerais MG Brazil) and two slideswere prepared for each participant

27 Laboratory Maintenance of the S mansoni Experimen-tal Cycle The S mansoni cycle was maintained throughperiodic infection of hamsters (Mesocricetus auratus) andBiomphalaria glabrata mollusks (strain BH) Each weekfive animals were subcutaneously infected with 200ndash300cercariae and sacrificed after 49 to 56 days to collect adultworms and parasite eggs S mansoni eggs were collected fromliver granulomas After washing with physiological solution


for complete removal of blood the worms and eggs werecounted and frozen

28 AdultWormTotal Extract Approximately 10000 Sman-soni adult worms of both sexes were thawed and resuspendedat a concentration of approximately 1000 wormsmL in10mM phosphate buffered saline (PBS) at pH 72 The pro-tease inhibitor phenylmethylsulfonyl fluoride (PMSF Sigma-Aldrich St Louis MO USA) was then added to producea final concentration of 1mM The suspension was groundin a manual tissue homogenizer in an ice bath for 1 hAfter thorough mixing the suspension was subjected totwo centrifugations at 10000timesg at 4∘C for 45min Thesupernatant was then removed and the protein content wasmeasured using the DC Protein Assay reagent (Detergent-Compatible Colorimetric Assay Kit Bio-Rad Hercules CAUSA) employing a modified Lowry method [24 25] Theextract was aliquoted and stored in a freezer at minus80∘C untilfurther use [26] This total extract was used for the sensitiza-tion of microplates to IgG (ELISA-IgG) antibodies [27 28]

29 TCA-Soluble Fraction A soluble fraction in trichloroace-tic acid (TCA-soluble fraction) was prepared from the crudeextract of adult worms of S mansoni according to a previ-ously described methodology [27] with modifications Anequal volume of 10 TCA was added to the total adult wormextract After mixing vigorously for three cycles of 1minusing a vortexer (Vortex Genie-2 Scientific Industries IncBohemia NY USA) the suspension was subjected to cen-trifugation at 10000timesg at 4∘C for 45min The supernatant(containing the TCA-soluble fraction) was removed andthen dialyzed against PBS on a cellulose membrane (Sigma-Aldrich) that retains substances with a molecular weight of12000 kDa or greater with continuous stirring overnight at4∘C We subsequently determined the protein content ofthe antigen solution (TCA-soluble fraction) which was thenaliquoted and stored in a freezer at minus80∘C until further useThe TCA-soluble fraction was used for the sensitization ofmicroplates to IgM (ELISA-IgM) antibodies [26 28]

210 Enzyme-Linked Immunosorbent Assays (ELISA) ForELISA-IgM polystyrene plates (Costar High Binding 3590Corning NY USA) were sensitized with 1 120583gwell of theTCA-soluble fraction of the S mansoni total extract dilutedin 01M carbonatebicarbonate buffer (pH 96) incubatingfor 2 h at 37∘C followed by 18 h at 4∘C in a humid chamberAfter washing three times with PBS containing 005Tween-20 (PBS-T) the plates were blocked for nonspecific sites with200120583L of 5 skim milk in PBS-T (PBS-TM 5) incubatingfor 2 h at 37∘C in a humid chamber The plates were thenwashed again with PBS-T Serum samples were diluted(1 100) in PBS-T with 2 skimmilk (PBS-TM 2) and testedin duplicate (50 120583Lwell) After incubation for 30min at 37∘Cin a humid chamber the plates were washed three timeswith PBS-T and 50120583L of a 120583-chain-specific anti-human IgMperoxidase conjugate (Sigma-Aldrich) diluted 1 5000 in PBS-TM 2 was added After 30min of incubation in a humidchamber the plates were washed with PBS-T and 100 120583L of

7

6

5

4

3

2

1

0

ELISA-IgG ELISA-IgMRe

activ

ity in

dex

Figure 2 Reactivity index of 612 sera obtained by ELISA-IgG andIgM-ELISA technique of the individuals in the city of Barra MansaRJ Brazil 2011

a chromogenic mixture consisting of tetramethylbenzidine(TMB) and H

2O2was added After 10min of incubation in

the dark the reactionwas quenchedwith 25120583L of 2NH2SO4

The absorbance at 450 nm was measured using an ELISAplate reader (Titertek Multiskan MCC340 Lab SystemsHelsinki Finland)

The reaction conditions described above for performingELISA-IgM testing were also used for ELISA-IgG testingexcept that polystyrene plates (Nunc PolySorp RoskildeDenmark) a 500 ngwell total antigen extract of S mansoniand an Fc-specific anti-human IgG peroxidase conjugate(Sigma-Aldrich) diluted 1 20000 were used

To determine the threshold of reactivity (cut-off) forELISA-IgG and ELISA-IgM receiver operating characteristic(ROC) curves were constructed We used 13 and 29 samplesfrom patients who tested positive and negative respectivelyfor S mansoni in the parasitological examination and IFTand 13 samples from patients who tested positive for otherhelminth parasites in the parasitological examination andnegative for S mansoni in IFT

The reactivity index (RI) of the samples was calculatedusing the equation IR = sample absorbancecut-off Serumsamples with IR ge 100 were considered reactive Figure 2

211 Detection of IgM Antibodies against Antigens of the Smansoni Digestive Tract IFT-IgM was used to detect anti-antigen polysaccharide IgM antibodies in the digestive tractof adult S mansoni worms on paraffin sections according tothe technique described by Silva et al [29] andNash 1974 [30]and 1978 [31]


212 Obtaining Adult Worms The infected hamsters wereanesthetized with an intramuscular injection of 100mgkg ofketamine and xylazine and sacrificed following the animalsacrifice guidelines of our institution After sectioning theportal vein 50mL of 085 saline solution with ethylenedi-aminetetraacetic acid (EDTA)was infused in the left ventricleusing a 20mL syringe through successive infusions by nothaving an infusion pump for maintaining a continuous flowAdult worms were obtained after perfusion of the portalsystem removed from the abdominal cavity and numberedand stored at minus20∘C Adult male worms were separated forldquoparticulaterdquo antigen processing in paraffin sections for IFT-IgM analysis

213 Slide Preparation for IFT-IgM Approximately 60 adultmale worms were placed on an end-jointed 200120583L meshscreen (Tecmolin PA-6-212XX Sao Paulo SP Brazil)immersed in Rossmanrsquos solution fixative for 2 h at roomtemperature and then immersed in 90 ethanol three timesfor 2 h The samples were then immersed in absolute alcoholfor 15 h and incubated in methyl benzoate for 4 h xylol at60∘C for 15min 50 xyleneparaplast for 15min and 100paraplast (Monjet Scient) at 60∘C for 30min The materialwas embedded in an L-shaped aluminum frame placed atroom temperature for 12 h and subsequently stored at thesame temperature for processing of histological sectionsSerial sections of 5 120583m thickness were cut using a microtomeat ten sections per slide

The slides containing paraffin sections were subjected todewaxing and rehydration using successive baths of xyleneand ethanol at different concentrations with a final bath inPBS pH = 72 The slides were stored at room temperatureuntil use

214 IFT-IgM Serum samples were diluted 1 10 in PBSsolution (pH 72) and deposited on the paraffined sections ofadult worms After incubation at 37∘C for 50min in a humidchamber slides were washed in 001M PBS (pH 72) bathsfor 10min Anti-human IgM fluorescent conjugate (goatanti-human IgG 120574-chain-specific fluorescein isothiocyanateantibody) was added (Sigma-Aldrich St Louis MO USA)in accordance with its optimal use titer (1320) in PBS pH =72 containing 1 Evans blue solution (Bio-Rad LaboratoriesWashington DCUSA) After further incubation andwashesslides were dried and mounted with glycerol and coverslipsPositive standard serum was used for 110 140 and 1160dilutions Negative standard serum was used for 110 dilu-tions

The IFT-IgM reading was performed using an OlympusBX-FLA fluorescence microscope (Olympus CorporationTokyo Japan) equippedwith an epi-illumination systemwith100x andor 200x magnification

A sample was considered positive when fluorescence waspresent only in structures related to the parasite digestive tract(Figure 3) Fluorescence detected in membranes or parasiteparenchyma was considered nonspecific The results wereexpressed as reactive (presence of fluorescence in the diges-tive tract) and nonreactive (absence of fluorescence) sera

Figure 3 Reactivity of the inner lining of the digestive tube positivehuman serum IgM antibodies

The classification of the sera was based on the fluorescenceintensity in the internal portion of the worms digestive tract(only male worms were used for embedment) according to atargeted fluorescence intensity scale The attribution of 1+ to4+ was obtained comparing the results with a photographicstandard built with serum nonreactive (0) low (1+) medium(2+) strong (3+) and very strong (4+)

215 Antibody Detection of S mansoni Egg Antigens Circu-moval precipitin test (COPT) was used to detect antibodyreactions against excretion and secretion products of Smansoni eggs using previously described techniques [32]

216 Isolation and Purification of S mansoni Eggs S mansonieggs were isolated and purified as described by Dresden andPayne [33] and Pinto et al [34] with some modifications

Livers from three infected hamsters were cut into smallpieces (3mm) and incubated in a water bath at 37∘C for20min in a solution containing 0004 pepsin and 07hydrochloric acid After incubation the peptic solution wasdiscarded and the tissue fragments were added to 150mLof 09 ice-cold saline solution containing 50 120583L of TritonX-100 The material was homogenized using several drivepulses in a domestic blender (Walita Philips AmsterdamNetherlands) until the fragments were completely rupturedThe material was then filtered in fourfold gauze and screenprocessed under negative pressure through a series ofmetallicsieves with mesh numbers 100 (0150mm) 200 (0075mm)and 400 (0038mm) (Granutest Telastem Peneiras ParaAnalise Ltda Sao Paulo SP Brazil)

Eggs retained on the last sievewere removed by successivewashing with ice-cold 09 saline solution and concentratedto 1mL volume by centrifugation at 154timesg for 15 sec Fromthis concentrated suspension a 10 120583L aliquot was placedbetween the blade and the coverslip and evaluated under amicroscope (Olympus Corporation Tokyo Japan) at 100xmagnification The number of eggs under the entire area


(a) (b) (c)

Figure 4 Intensity patterns of RPO (a) RPO pattern 1 (b) RPO pattern 2 (c) RPO pattern 3

of the coverslip was counted Suspensions exceeding 20cell debris in relation to the number of eggs found werereprocessed through the 200 and 400mesh sieves For COPTthe egg suspensions were adjusted to contain 300 viable eggsper 10 120583L of 09 saline solution and stored at 4∘C for 4 h untilreaction preparation

217 Circumoval Precipitin Test Aliquots (10 120583L) of eachpurified egg suspension adjusted to contain 300 viable eggsin 09 saline solution and 50 120583L of each serum sample wereadded to 2mL Eppendorf tubes (Eppendorf do Brasil LtdaSao Paulo SP Brazil) and incubated at 37∘C for 48 h Subse-quently a 30 120583L aliquot of each mixture (serum + viable eggsuspension)was placed on a slide and coveredwith a coverslip(22 times 22mm) Assessment was performed on a binocularOlympus-CX41 microscope (Olympus Corporation TokyoJapan) with a 10x or 40x objective lens The number ofreactive eggs per 100 viable eggs and the periovular precipitatemorphology were used for assessment S mansoni eggs wereconsidered reactive if they contained globular precipitates ofvariable size and form or if they appeared as small or longseptated chains similar to Taenia segments (Figure 4) Serawere considered positive if at least 9 of themature eggs werereactive in the presence of different precipitates [35]

218 TaqManReal-Time PCRAssays Preanalysis preparationof feces samples for qPCR Fecal samples without conserva-tives were passed through a Nylon Baltex PA-7-200XX filter(Tecmolin Sao Paulo SP Brazil) to remove larger impuritiesUsing amarked stainless steelmeasuring plate approximately500mg of feces from each sample was aliquoted and stored ina freezer at minus20∘C

219 DNA Extraction from Serum DNA was extracted fromhamster serum samples and the 09 saline solution contain-ing 200 eggsmL using the guanidine isothiocyanate-phenol-chloroform (GT) method [36 37] DNA was stored at minus20∘Cafter extraction

220 DNA from Fecal Samples DNA extraction from ham-ster feces was performed in two phases in the first phaseafter resuspending approximately 500mg of stool in 1mL01M PBS five glass beads were added This mixture washomogenized for 5min using a vortex mixer followed bycentrifugation for 8min at 16863timesg at 4∘C An aliquotof 400 120583L of the supernatant was mixed with 100120583L ofRapid One-Step Extraction (ROSE) solution 10mM Tris-hydroxymethyl amino methane HCl pH 8 300mM EDTApH 80 1 sodium lauroyl sarcosinate (Sarkosyl) and 1polyvinylpolypyrrolidone (PVPP) [38] Then 30 120583L of Pro-teinase K (Life Technologies Carlsbad CA USA) was addedand homogenized using a vortex mixer The sample wasincubated for 120min at 65∘C In the second phase the DNApresent in this solution was extracted and stored as describedfor serum samples

221 Purification of DNA Extracted from Fecal and SerumSamples DNA extracted from serum and feces at distincttimes after infection was purified using the InstaGeneMatrixin accordance with the manufacturerrsquos instructions Oncepurified extracted DNA was stored at minus20∘C until use

222 Amplification of DNA from Serum and Fecal Samples

Primers and Probes A set of primers and probes com-plementary to a 121 bp tandem repeat sequence from Smansoni strain SM 1-7 (GenBank accession numberM61098)described by Hamburger et al [39] were used for amplifi-cation and detection of S mansoni DNA Primer sequenceswere as follows forward F2 51015840-CCG ACC AAC CGT TCTATG A-31015840 reverse R2 51015840-CAC GC TCT CGC AAA TAATCT AAA-31015840 probe PO2 51015840-6[FAM] TCG TTG TAT CTCCGA AAC CAC TGG ACG [(BHQ1])-31015840 all probes weresynthesized by Sigma Life Sciences (Woodlands TX USA)

All samples were evaluated using TaqMan ReagentsExogenous Internal Positive Control (IPC) (Life Technolo-gies) in accordance with the manufacturerrsquos instructions totest for the presence of Taq DNA polymerase inhibitors


For the positive control of the qPCR-feces in all assayswe used DNA obtained in the extraction of 200 eggsmL ofsaline 09

223 TaqMan Real-Time PCR Conditions for Serum and FecalSamples TaqMan Real-Time PCR was performed in a finalvolume of 20 120583L containing 10 120583L TaqMan Universal PCRMaster Mix 2X 20 pmol of primers F2 and R2 5 pmol ofthe PO2 probe and 2 120583L of purified DNA For each sampleanother reactionwas performed in parallel using the TaqManReagents Exogenous Internal Positive Control (IPC) in afinal volume of 21120583L containing 10 120583L of TaqMan UniversalPCR Master Mix 2X 5 120583L 10X Exogenous IPC mix 1120583L50X Exo IPC and 5 120583L of purified DNA samples For eachbatch of reactions two additional controls were used ano amplification control (NAC) and a no template controltarget (NCT) PCR was performed in an Applied Biosystems7300 Real-Time PCR System (Life Technologies) using thefollowing cycling conditions 50∘C for 2min 95∘C for 10minand 40 cycles at 95∘C for 15 sec and 60∘C for 1min

To detect S mansoni the Applied Biosystems 7300 Real-Time PCR System standard cycles were used 50∘C for 2min95∘C for 10min 40 cycles at 95∘C for 15 sec and 60∘C for1min

For the positive control of the qPCR-serum in all assayswe used DNA obtained in the extraction of 200 eggsmL ofsaline 09

Furthermore the possibility of contamination was min-imized by performing DNA extraction and amplificationin separate rooms by performing all experiments inside alaminar flow cabinet by frequent use of ultraviolet (UV) (BioII A-Telstar Life Science Vancouver Canada) irradiationand by using only disposable sterile laboratory equipmentand pipette tips with filters

224 Criteria Used to Evaluate the Results of qPCR UsingStool and Serum Samples The following criteria were usedto evaluate the qPCR

Positive qPCR duplicates of the samplewere amplifiedby qPCRUndetermined qPCR one aliquot of the sample wasamplified by qPCRqPCR IPC exogenous control for the testing samplewas not amplified (negative) by qPCR

The undetermined qPCR and qPCR IPC samples weretested in triplicate Duplicate amplifications of the samplesobtained by qPCR were included in the positive results [38]

3 Results

The characteristics of the sample population (650 freely par-ticipating individuals) residing in the five suburbs of the cityof Barra Mansa (RJ) used to evaluate the techniques ELISA-IgG ELISA-IgM ITF-IgM COPT qPCR-serum qPCR-fecesKK and HH are shown in Table 1

Analysis of Table 1 reveals that the majority of the indi-viduals were female with an average age of 397 The greatest

Table 1 Sociodemographic characteristics of the study populationindividuals from the city of Barra Mansa RJ 2011

Characteristic Frequency SexFemale 385 592Male 265 408

Age group (years)1 to 9 27 4210 to 19 144 22220 to 49 250 38550 or over 229 352Average age (SD) 397 (211)

LiteracyYes 624 960No 22 34Not reported 4 06

NeighborhoodCantagalo 47 72Nova Esperanca 187 288Santa Clara 35 54Sao Luiz 102 157Siderlandia 279 429

Water supplyGeneral network 551 848Well or spring 79 122Others 1 02Not reported 19 29

Use of river waterNo 473 728Washing clothes 15 23Washing utensils 2 03Baths 5 08Swimming 5 08Sand extraction 7 11Not reported 143 220

Destination of feces and urineSewer system 486 748Septic tank 7 11In the open 117 180Not reported 40 62

Previous schistosomiasisYes 25 38No 519 798Not reported 106 163

number of participants resided in the neighborhood of Sin-derlandia (429) and approximately 4 of the participantsreported a history of schistosomiasis

The positivity for infection by S mansoni according toeach diagnostic technique used in this study is shown inTable 2

The technique that presented most evidence of infec-tion by S mansoni was the ELISA-IgM (214) whereas


Table 2 Positivity for infection by S mansoni according todiagnostic technique in samples collected from individuals in thecity of Barra Mansa RJ 2011

Technique Positivetotal KK-HH 5610 08ELISA-IgG 71612 116ELISA-IgM 131612 214COPT 33612 54ITF-IgM 97612 158qPCR-feces 60610 98qPCR-serum 9612 15

Table 3 Prevalence of schistosomiasis mansoni and otherenteroparasites as determined using the KK and HH techniques inindividuals from the city of Barra Mansa RJ 2011

Parasitosis HHKK Frequency Schistosoma mansoni 5 08Endolimax nana 106 174Entamoeba coli 28 46Entamoeba hartmanni 1 02Entamoeba histolyticadispar 5 08Blastocystis spphominis 66 108Giardia lambliaintestinalis 11 18Iodamoeba butschlii 1 02Enterobius vermicularis 6 10Strongyloides stercoralis 9 15Ascaris lumbricoides 4 07Trichuris trichiura 3 05Taenia sp 2 03

the parasitological techniques (KK and HH) showed thelowest rates of infection (08)

In the KK technique the quantity of eggs detected pergram of feces varied from 0 to 456 eggs One of the fivepositive cases was diagnosed only with the HH technique

The prevalence of infection with S mansoni and variousenteroparasites diagnosed by the KK and HH techniques inindividuals from the city of BarraMansaRJ 2011 is shown inTable 3

Of the individuals tested 279 were found to be infectedwith at least one parasite

The results of the KK technique showed low positivity forany diagnosed parasitosis and the positivity rate for infectionby S mansoni was 07 (119899 = 4)

The ELISA-IgG reactivity index showed an average of212 with an SD of 110 (median = 185 minimum = 101maximum = 672 119899 = 71) The ELISA-IgM reactivity indexshowed an average of 224 with an SD of 109 (median = 191minimum = 102 maximum = 467 119899 = 131) The IFT-IgMpositivity showed an average of 156 crosses with an SD of069 (median = 1 minimum = 1 maximum = 3 119899 = 97)COPT reactivity was observed in an average of 21 of viableeggs with an SD of 9 (median 21 viable eggs minimum =9 viable eggs maximum 38 viable eggs 119899 = 33)

Table 4 shows that the results of the qPCR technique thatwere positive (Figure 6) and undetermined occurred with afrequency of 98 and 89 and a median of 348 and 371respectively

In this study we found that in 162 (119899 = 99) of the fecalsamples amplification of the exogenous control (IPC) and Smansoni DNA was unsuccessful

In serum samples (Table 5) the Ct values of the qPCRpositive (Figure 6) and undetermined samples were 15 (119899= 9) and 51 (119899 = 31) and the median was 363 and 370respectively

Table 6 shows that the positivity rate of the COPTtechnique differed significantly from all other techniques itshowed a lower positivity rate than the ELISA-IgG ELISA-IgM IFT-IgM (119875 lt 0001) and qPCR-feces (119875 = 0001)techniques and a higher positivity rate than the KK HHand qPCR-serum techniques (119875 lt 0001) The results of theCOPT technique presented greater concordance with thosefrom ELISA-IgG (Kappa = 0377) IFT-IgM (Kappa = 0347)and qPCR-feces (Kappa = 0311) techniques

When comparing COPT with the other diagnostic tech-niques ELISA-IgM assay presented the highest sensitivity(Table 7)

4 Discussion

Diagnosis of S mansoni is challenging and determining theprevalence of infection is therefore difficult Schistosomiasisis a chronic infection that may not progress to a severe formbut that can trigger debilitating sequelae that are secondaryto parasitism This is particularly true in children whomay experience delayed growth and develop anemia andchronic malnutrition [2] Moreover asymptomatic carrierscan potentially transmit schistosomiasis in areas that lackadequate sanitation [40]

Because of the present state of endemism of schistoso-miasis in Brazil the current proposal is the interruption(elimination) of its transmission Thus there is a need todevelop new techniques that are more sensitive and specificto permit early diagnosis and treatment of infected individu-als necessary for the control of this helminthiasis [3]

In this studywe compared the performance of theKKandHH techniques the ELISA-IgG ELISA-IgM and the ITF-IgM techniques as well as qPCR of stool and serum samplesusing the COPT technique as a reference [19 20 41]

The positivity rate for schistosomiasis in our study popu-lation as determined by immunological techniques rangedfrom 214 (131612) for ELISA-IgM to 158 (97612) forITF-IgM and 116 (71612) for ELISA-IgG These rates areabove those for parasitological techniques to diagnose Smansoni infection The low sensitivity of the parasitologicalmethod in ALEs had previously been described [13ndash18 2842 43]

The high positivity rates observed in this study for theELISA-IgG ELISA-IgM and ITF-IgM techniques could havebeen caused by inclusion of individuals previously infectedby S mansoni but properly treated and cured or inclusionof those that were exposed to very small loads of cercariae


Table 4 Results from the positive and undetermined qPCRs with the threshold cycle (Ct) values in fecal samples from the population ofthe city of Barra Mansa RJ 2011

Technique Casestotal Ct values (LOG) qPCRAverage SD Median Minimum Maximum

qPCR positive 60610 98 336 49 348 147 388qPCR undetermined 54610 89 323 85 371 115 400

Table 5 Description of the threshold cycle (Ct) for the results from the positive and undetermined qPCR in serum samples from theindividuals sampled from the city of Barra Mansa RJ 2011

Sample Technique Casestotal Ct values (LOG) qPCRAverage SD Median Minimum Maximum

Serum qPCR positive 9612 15 369 13 363 388 352qPCR undetermined 31612 51 355 44 37 397 203

Table 6 Concordance between the positive results obtained using the COPT technique and those obtained using the other techniques infecal and serum samples collected from the sample population in the city of Barra Mansa RJ 2011

TechniquesCOPT Total IC (95)

Negative Positive 119875McNemar Kappa119899 () 119899 () 119899 () Lower Higher

KK-HHNegative 542 (948) 25 (44) 567 (991)

lt0001 0224 0038 0409Positive 1 (02) 4 (07) 5 (09)Total 543 (949) 29 (51) 572 (1000)

ELISA-IgGNegative 530 (866) 11 (18) 541 (884)


ELISA-IgMNegative 475 (776) 6 (1) 481 (786)


IFT-IgMNegative 508 (830) 7 (11) 515 (842)


qPCR-fecesNegative 503 (879) 14 (24) 517 (904)

0001 0311 0176 0446Positive 40 (70) 15 (26) 55 (96)Total 543 (949) 29 (51) 572 (1000)

qPCR-serumNegative 574 (938) 29 (47) 603 (985)


or unisexual infections [16 44 45] The possibility of cross-reactivity with cercariae antigens from parasites that infectother animal species and cross-reactivity with other parasitesmust also be considered [28 46ndash48]

The positivity rate of the COPT technique also differedsignificantly from the positivity rates of the parasitologicaltechniques (119875 lt 0001) The level of concordance (119896 =0224) for these techniques was higher than that observed for

the other immunodiagnostic techniques (ELISA-IgG ELISA-IgM and ITF-IgM)

This higher positivity rate observed for the COPT couldbe due to the fact that oviposition occurs with egg depositionin the deeper layers of the intestine evenwhen eggs are absentfrom the feces mainly in chronic infections in patients over40 years of age and in schistosomiasis infections with lowparasite load [49 50]


Table 7 Description of the sensitivity specificity the positive likelihood ratio the negative likelihood ratio the positive predictive value andthe negative predictive value of all diagnostic techniques compared to the COPT technique in individuals from the city of Barra Mansa RJ2011

Techniques Parameters Estimate IC (95)Lower Higher

KK-HH

Sensitivity () 138 39 317Specificity () 998 990 1000Likelihood ratio (+) 749 86 6490Likelihood ratio (minus) 09 07 10Positive predictive value (PPV) () 800 284 995Negative predictive value (NPV) () 956 936 971Accuracy () 955 951 958

ELISA-IgG


ELISA-IgM


IFT-IgM


qPCR-feces


qPCR-serum



The COPT positivity was lower than that for the ELISA-IgG and ELISA-IgM and ITF-IgM tests probably as aresult of the different nature of the antigens The COPTdetects antibodies against excretion products and secretionsof miracidia or antibodies against antigens present in thefluid surrounding S mansoni eggs whereas the ELISA-IgG ELISA-IgM and ITF-IgM techniques detect antibodiesagainst antigens in the internal coating of the digestive tubeof adult worms [32 50]

The appearance of the antibodies detected by the COPT isprecocious and coincides with the beginning of the elimina-tion of the eggs in the feces [49 51 52] Thus the antibodiesdetected by the COPT are only produced when worms ofboth sexes cause the infection the ELISA-IgG ELISA-IgMand ITF-IgM techniques can also detect unisexual infections[44 52 53]

To the best of our knowledge this is the first time thatqPCR-feces and qPCR-serumwere used in a population studyin low endemicity area for S mansoni infection

The qPCR-feces technique exhibited 12-fold higher pos-itivity than the KK and HH techniques at 98 (119899 = 60)compared to 08 (119899 = 5) for the parasitological techniquesThe qPCR-feces positivity rate was only higher than the ratesfor the COPT (54) and the qPCR-serum (15) techniquesits results differed significantly from all techniques (119875 lt 005)except the ELISA-IgG technique

The positivity rate of the qPCR-serum technique washigher than those of the parasitological techniques but lowerthan those of the other techniques including the qPCR-fecestechnique A study by Pontes et al [23] also indicated thatthe qPCR-serum technique has lesser sensitivity than theconventional PCR-feces technique for detecting S mansoniinfection HoweverWichmann et al [54] and Zhou et al [55]correlated the positivity in the acute phase of schistosomiasisinfection with inflammatory alterations The acute phasewould cause increased circulation of schistosomiasis degra-dation products both adult worms and eggs thus increasingthe quantity of circulating DNA This phase would usuallycontinue until the eighth week after infection and thesedata would explain the higher rate of amplification using theqPCR-serum technique

The median of the Ct values of the logarithmic curvesfor the qPCR-feces and serum techniques was 348 and363 respectively indicating low amplification of the DNAtemplate found in egg fragments (002 eggs at a dilution of110000) based on the standardization of the technique Thelow parasite load in S mansoni infections observed in ALEscould explain this result [56]

Wichmann et al [57] reported a higher positivity rate forthe qPCR-serum technique in patients with acute schistoso-miasis using the presence of S mansoni DNA in duplicatesamples as a criterion for positivity with a limit of Ct lt 45

In spite of the presence of factors that inhibit the qPCR-feces technique and the low parasite load in the sampledpopulation the qPCR-feces technique showed a 12-foldhigher positivity rate (60610) than the KK and HH (5610)techniques the qPCR-serum technique presented a positivity(9612) of approximately twofold higher than those of theparasitological techniques

According to Cnops et al [58] all PCR-based techniquesfor genomic DNA of S mansoni were able to detect the DNAfrom all phases of the life cycle of this parasite eggs contain-ingmiracidia cercariae schistosomulae and adult worms Insummary a positive PCR assay indicates the presence of theparasite but does not provide information regarding its lifecycle phase or its viability including the presence of maturemale and female worms capable of egg deposition This islikely to explain why we have not found a positive correlationbetween the Ct value and the number of eggs

The technique developed by Kato and Miura [10] mod-ified by Katz et al [59] became the international referencetechnique for diagnosing schistosomiasis infection [60]However a decline in the sensitivity of this technique wasobserved in individuals with low parasite load infectionsresiding in ALEs following specific treatment which com-promises the evaluation of new techniques for diagnosinginfection by S mansoni when it is used as a reference [61 62]

Various studies have been performed to identify alter-native methods of diagnosis applicable on a large scalethat could be used to aid the programs for epidemiologicalvigilance in areas where despite control actions new cases ofconfirmed autochthony still occur [63]

Immunodiagnostic techniques are still used in schisto-somiasis control programs in countries such as China andVenezuela [43 50 64 65] In Brazil various techniques havebeen tested in regions considered to have low endemicitysuch as in the states of Sao Paulo and Rio de Janeiro [28 4048 66ndash68]

The COPT is considered by some researchers to be thegold-standard technique for diagnosing schistosomiasis inALEs [69 70]

Taking into account the possibility of employing par-asitological and immunological techniques for diagnosingschistosomiasis infection in ALEs Alarcon deNoya et al [50]proposed three laboratory criteria for case definition in theseareas

(I) individuals eliminating S mansoni eggs in feces Inthis group COPT and serological positivity reactionsare common

(II) individuals without S mansoni eggs in their feceswith positive COPT who have not received specifictreatment in the previous 12 months Usually thisgroup shows positivity for one andor two serologicaltechniques

(III) individuals without S mansoni eggs in their feceswith negative COPT who have not received specifictreatment in the previous 12 months but who haveshown positivity for two serological techniques

Use of criteria (I) and (II) presented above would yield34 positive cases in our study population and the prevalencewould be approximately 56 (34612) greater than theprevalence detected by the parasitological techniques 08(5612) prevalence detected by the parasitological techniquesused in the program for schistosomiasis vigilance

As we verified in this study various techniques could beused for diagnosis of schistosomiasis however all present


problems of sensitivity or specificity Thus we could notidentify in this study the ideal technique that combineshigh rates of sensitivity and specificity and low cost andeasy applicability even in the field probably due to thelow prevalence of infection (approximately 1) in the areastudied All assays employed showed low sensitivity andspecificitywhen applied to the study population Lowparasiteload very recent infections and the low number of positiveparasitological exams may explain this

One limitation of this study is the collection of a singlefecal sample per individual as recommended by the guide-lines of the National Program for Schistosomiasis since thesensitivity of the KK test could have been improved bytesting samples collected on two or three successive daysto compensate for daily variation in egg production in aninfected individual

The present study indicates that the use of combinedtools can improve the diagnosis of low parasite load Smansoni infections in both clinical and epidemiologicalstudies The benefits of parasitological techniques for thecontrol of S mansoni are widely recognized particularly theKK technique We consider the presence of ALEs to be anindicator of the positive outcome of schistosomiasis controlprograms worldwide However in order to eliminate the riskof schistosomiasis in populations of low endemicity areasthe pursuit of novel techniques must be considered Poten-tially effective methods may include novel immunodiagnos-tic techniques molecular biology tools and parasitologicaltechniques such as saline gradient andmagnetic bead-basedisolation of eggs [26 37 71ndash75] The development of thesetools and the subsequent elimination of schistosomiasis willbe a great challenge that will require the combined efforts ofgovernments health professionals and researchers

To advance this agenda we suggest a change to the thirdcriterion proposed byAlarcon deNoya et al [50] consideringcases of schistosomiasis in which individuals present positiveqPCR-serum andor qPCR-feces results We withdrew thecriterion regarding positivity in two serological techniquesand negative results in the parasitological and the COPTtechniques (Table 8)

With regard to field activities we propose serological andparasitological survey of ALEsWe recommend the use of theELISA-IgM and ELISA-IgG techniques for an initial triageof the target population due to their greater sensitivity tothe possibility of automation and to their ability to providequantitative results We suggest the use of the KK and HHparasitological techniques for evaluating the intensity of Smansoni infections and identifying the presence of otherparasitic infections respectively

Following the triage we recommend that actions bedetermined according to laboratory classification criteriaThus individuals classified under criteria (I) (II) and (III)would be referred for clinical evaluation specific chemother-apy and health education For individuals with positiveresults for one andor two serological techniques (ELISA-IgG or ELISA-IgM) we propose performing the COPT Forthe patients who present a positive COPT we recommenda clinical evaluation treatment and health education If theresult of the COPT is negative we recommend performing

Table 8 Proposal for laboratory criteria for defining cases ofschistosomiasis in ALEs

Criterion I Criterion II Criterion IIIIndividualseliminating Smansoni eggs infeces

Positive COPT Individuals with no Smansoni eggs in feces

Withwithoutpositive COPT

Individuals with noS mansoni eggs infeces

Negative COPT

WithwithoutpositiveELISA-IgM andorELISA-IgGtechniques

Without specifictreatment in the last12 months

Withwithoutpositive ELISA-IgMandor ELISA-IgGtechniques

Positive qPCR-serumandor positiveqPCR-feces

qPCR-feces and qPCR-serum if any of these molecular tech-niques yield a positive result we suggest clinical evaluationspecific treatment and health education (Figure 5)

We propose the combination of the ELISA-IgM andIgG techniques in the triage phase due to the possibilityof automation and to their higher levels of sensitivity thusenabling the identification of a greater number of casesIn addition the ELISA-IgM technique is more sensitiveand the ELISA-IgG technique is more specific hence theircombination will improve diagnosis

According to our proposal positivity of one or twoimmunological techniques indicates the need for confirma-tion by the COPT which despite being more difficult toperform can identify active infections or by the molecularreactions which can identify the presence of the parasite atany stage of infection

The possibility of the use of rapid tests with recombinantantigens [76] or with Circulating Anodic Antigen (CAA)in urine feces or serum [77ndash80] might be an interestingalternative for field works in places where health workersdo not have the proper training and equipment to performsophisticated serological assays

However continued use of the parasitological techniqueswill be important in particular the HH technique as itpermits the evaluation of the parasite load in positive casesand allows observation of different levels of prevalence aswell Besides the evaluation and treatment the infection riskfactors must be taken into account

We believe that maintaining this level of surveillancein ALEs will present a great challenge and will requireevaluation of costs interactionswith other studies and finallya broad discussion involving the various actors in the contextof public health research and society

In Brazil recent epidemiological data on S mansonipoint to an increase in ALEs and the current proposal aimsto interrupt the transmission of this helminth (Ordinancenumber 2556 of 28 October 2011) In this context diagnosisof S mansoni becomes a strategic hurdle we must overcome


Individualsclassified by

criteria I II and III

Clinicalevaluation

specificchemotherapy and

health education

Clinicalevaluation


health education

Clinical evaluationspecific

chemotherapy andhealth education

ELISA-IgM andor ELISA-IgG

COPT

Positive Negative

qPCR-feces andorqPCR-serum

Positivity in any of themolecular techniques

(I) Serological ELISA-IgM and ELISA-IgG and Coproscopic (KK-HH) inquiry(II) Case definition according to laboratory classification criteria

S mansoni infection risk factors

Figure 5 Proposal for epidemiological vigilance in ALEs

10

15

20

25

30

35

40

45

Feces (positives) Serum (positives)

Ct

Figure 6 Description of the threshold cycle (Ct) for the resultsfrom the positive and qPCR in serum and feces samples from theindividuals sampled from the city of Barra MansaRJ 2011

in order to reach the desired result indicators in the process ofeliminating this parasitic infection In addition the develop-ment of new diagnostic laboratory techniques for detectinglow parasite load infections represents an important strategyto overcome this need in clinical contexts as well as in publichealth

Conflict of Interests

The authors declare that they have no competing interests

Authorsrsquo Contribution

Maria Cristina Carvalho do Espırito-Santo Monica VivianaAlvarado-Mora Pedro Luiz Silva Pinto Expedito Jose deAlbuquerque Luna and Ronaldo Cesar Borges Gryschekconceived and designed the experiments Maria CristinaCarvalho do Espırito-SantoMonicaVivianaAlvarado-MoraEmmanuel Dias-Neto Pedro Luiz Silva Pinto Maria Car-men Arroyo Sanchez Joao Renato Rebello Pinho Vera

Lucia Pagliusi Castilho and Elenice Messias do NascimentoGoncalves performed the experiments Maria Cristina Car-valho doEspırito-Santo JoaoRenatoRebello Pinho Flair JoseCarrilho Expedito Jose de Albuquerque Luna EmmanuelDias-Neto and Ronaldo Cesar Borges Gryschek contributedwith reagentsmaterialanalysis tools Maria Cristina Car-valho do Espırito-Santo Monica Viviana Alvarado-MoraExpedito Jose de Albuquerque Luna Maria Carmen ArroyoSanchez Pedro Luiz Silva Pinto Joao Renato Rebello PinhoEmmanuel Dias-Neto and Ronaldo Cesar Borges Gryschekwroteedited the paper All authors read and approved thefinal paper

Acknowledgments

This study was supported by the Sao Paulo Research Foun-dation (Grants 0753457-7 0850461-6 and 1052615-0)The authors would also like to thank Ms Maria CristinaConceicao de Mello (LIM-06-FMUSP) for her excellenttechnical assistance concerning the maintenance of the Smansoni biological cycle Emmanuel Dias-Neto and JoaoRenato Rebello Pinho are research fellows of the Con-selho Nacional de Desenvolvimento Cientıfico e Tecnologico(CNPq) Finally theywould like to thankDrWiltonNeri andstaff of theMunicipalHealthDepartment of BarraMansa Riode Janeiro Brazil

References

[1] L Chitsulo D Engels A Montresor and L Savioli ldquoThe globalstatus of schistosomiasis and its controlrdquo Acta Tropica vol 77no 1 pp 41ndash51 2000

[2] WHO Schistosomiasis Fact Sheet no 115 World Health Organi-zation Geneva Switzerland 2014 httpwwwwhointmediac-entrefactsheetsfs115en

[3] Ministry of Health Department of Health Surveillance Infor-mation System of Schistosomiasis Control Program and Dis-eases Information System Confirmed Cases of SchistosomiasisBrazil Major Regions and Federative Units From 1995 to 2011


Ministry of Health Department of Health Surveillance Infor-mation System of Schistosomiasis Control Program DiseasesInformation System Brasılia Brazil 2012

[4] N R Bergquist ldquoSchistosomiasis from risk assessment tocontrolrdquo Trends in Parasitology vol 18 no 7 pp 309ndash314 2002

[5] L C Pordeus L R Aguiar L RM Quinino and C S BarbosaldquoA ocorrencia das formas aguda e cronica da esquistossomosemansonica no Brasil no perıodo de 1997 a 2006 uma revisao deliteraturardquo Epidemiologia e Servicos de Saude vol 17 no 3 pp163ndash175 2008

[6] WHO ldquoPrevention and control of schistosomiasis and soil-transmitted helminthiasis report of a WHO expert committeeon the Control of Schistosomiasisrdquo Technical Report Series 912WHO Geneva Switzerland 2002

[7] Ministry of Health Health Surveillance Secretariat Depart-ment of Analysis of Health Situations Mortality InformationSystem Deaths from Schistosomiasis Major Regions and Federa-tive Units 1990 to 2011 Ministry of Health Brasılia Brazil 2012

[8] State Department of Health and Civil Defense and Governmentof the State of Rio de Janeiro Health Information Booklet of theState of Rio de Janeiro Government of the State of Rio de JaneiroRio de Janeiro Brazil 2009

[9] Municipal Health Secretariat and Epidemiology Coordina-tion Office ldquoDocuments taken from SINANNET07-05-2008rdquoBarra Mansa Rio de Janeiro Brazil 2008

[10] K Kato and M Miura ldquoComparative examinationsrdquo JapaneseJournal of Parasitology vol 3 p 35 1954

[11] L K Martin and P C Beaver ldquoEvaluation of Kato thick-smeartechnique for quantitative diagnosis of helminth infectionsrdquoTheAmerican Journal of Tropical Medicine and Hygiene vol 17 no3 pp 382ndash391 1968

[12] W A Hoffman J A Pons and J L Janer ldquoThe sedimentation-concentration method in schistosomiasis mansonirdquoThe PuertoRico Journal of Public Health and Tropical Medicine vol 9 pp283ndash291 1934

[13] E Ruiz-Tiben G VHillyerW B Knight I Gomez de Rios andJ P Woodall ldquoIntensity of infection with Schistosoma mansoniits relationship to the sensitivity and specificity of serologictestsrdquo American Journal of Tropical Medicine and Hygiene vol28 no 2 pp 230ndash236 1979

[14] A Sleigh R Hoff K Mott et al ldquoComparison of filtrationstaining (Bell) and thick smear (Kato) for the detection andquantitation of Schistosoma mansoni eggs in faecesrdquo Transac-tions of the Royal Society of Tropical Medicine and Hygiene vol76 no 3 pp 403ndash406 1982

[15] M L Barreto D H Smith and A C Sleigh ldquoImplications offaecal egg count variation when using the Kato-Katz methodto assess Schistosoma mansoni infectionsrdquo Transactions of theRoyal Society of Tropical Medicine and Hygiene vol 84 no 4pp 554ndash555 1990

[16] S J de Vlas and B Gryseels ldquoUnderestimation of Schistosomamansoni prevalencesrdquo Parasitology Today vol 8 no 8 pp 274ndash277 1992

[17] B Gryseels and S J de Vlas ldquoWorm burdens in schistosomeinfectionsrdquo Parasitology Today vol 12 no 3 pp 115ndash119 1996

[18] A Ebrahim H El-Morshedy E Omer S El-Daly and RBarakat ldquoEvaluation of the Kato-Katz thick smear and formolether sedimentation techniques for quantitative diagnosis ofSchistosoma mansoni infectionrdquoThe American Journal of Trop-ical Medicine and Hygiene vol 57 no 6 pp 706ndash708 1997

[19] G V Hillyer E Ruiz Tiben W B Knight I Gomez de Riosand R P Pelley ldquoImmunodiagnosis of infection with Schisto-soma mansoni comparison of ELISA radioimmunoassay andprecipitation tests performed with antigens from eggsrdquo TheAmerican Journal of Tropical Medicine and Hygiene vol 28 no4 pp 661ndash669 1979

[20] Y Zhu W He Y Liang et al ldquoDevelopment of a rapid simpledipstick dye immunoassay for schistosomiasis diagnosisrdquo Jour-nal of Immunological Methods vol 266 no 1-2 pp 1ndash5 2002

[21] DGAltmanPractical Statistics forMedical Research Chapmanamp Hall London UK 1991

[22] Her Majestyrsquos Stationery Office (HSMO) and Office of PublicSector Information (OPSI) Geneva Conventions (Amendment)Act 1995 (c 27) HerMajestyrsquos Stationery Office (HSMO) Officeof Public Sector Information (OPSI) 1995

[23] L A Pontes M C Oliveira N Katz E Dias-Neto and ARabello ldquoComparison of a polymerase chain reaction and theKato-Katz technique for diagnosing infection with Schistosomamansonirdquo The American Journal of Tropical Medicine andHygiene vol 68 no 6 pp 652ndash656 2003

[24] O H Lowry N J Rosebrough A L Farr and R J RandallldquoProtein measurement with the Folin phenol reagentrdquo TheJournal of Biological Chemistry vol 193 no 1 pp 265ndash275 1951

[25] G L Peterson ldquoReview of the Folin phenol protein quantitationmethod of Lowry Rosebrough Farr and Randallrdquo AnalyticalBiochemistry vol 100 no 2 pp 201ndash220 1979

[26] M C Espirito-Santo M C Sanchez A R Sanchez et alldquoEvaluation of the sensitivity of IgG and IgMELISA in detectingSchistosoma mansoni infections in a low endemicity settingrdquoEuropean Journal of Clinical Microbiology amp Infectious Diseasesvol 33 no 12 pp 2275ndash2284 2014

[27] A M Deelder H T M Klappe G J M J van den Aard-weg and E H E M van Meerbeke ldquoSchistosoma mansonidemonstration to two circulating antigens in infected hamstersrdquoExperimental Parasitology vol 40 no 2 pp 189ndash197 1976

[28] E J de Oliveira H Y Kanamura and D M Correia LimaldquoEfficacy of an enzyme-linked immunosorbent assay as adiagnostic tool for schistosomiasis mansoni in individuals withlow worm burdenrdquo Memorias do Instituto Oswaldo Cruz vol100 no 4 pp 421ndash425 2005

[29] R M Silva M I P G Silva S A G Vellosa E T Garciaand H Y Kanamura ldquoPesquisa de anticorpos IgM contra tubodigestivo do verme para prognostico da esquistossomosemansonicardquo Revista Brasileira de Patologia Clınica vol 28 pp39ndash42 1992

[30] T E Nash ldquoLocalization of the circulating antigen withinthe gut of Schistosoma mansonirdquo American Journal of TropicalMedicine and Hygiene vol 23 no 6 pp 1085ndash1087 1974

[31] T E Nash ldquoAntibody response to a polysaccharide antigenpresent in the schistosome gut I Sensitivity and specificityrdquoTheAmerican Journal of Tropical Medicine and Hygiene vol 27 no5 pp 939ndash943 1978

[32] J OLIVER-GONZALEZ ldquoAnti-egg precipitins in the serum ofhumans infected with Schistosoma mansonirdquo The Journal ofinfectious diseases vol 95 no 1 pp 86ndash91 1954

[33] M H Dresden and D C Payne ldquoA sieving method for thecollection of schistosome eggs from mouse intestinesrdquo Journalof Parasitology vol 67 no 3 pp 450ndash452 1981

[34] P L S Pinto L D Floriano S C Ferreira L M Sutoand S A G Vellosa ldquoPurificacao de ovos de Schistosomamansoni a partir de vısceras de hamsters (Crycetus auratus)


experimentalmente infectadosrdquo in XIV Congresso da SociedadeBrasileira de Parasitologia vol 23 p 264 Revista de PatologiaTropical Goiania Brazil 1995

[35] O Noya B Alarcon de Noya S Losada et al ldquoLaboratorydiagnosis of schistosomiasis in areas of low transmission areview of a line of researchrdquo Memorias do Instituto OswaldoCruz vol 97 no 1 pp 167ndash169 2002

[36] P Chomczynski and N Sacchi ldquoSingle-step method of RNAisolation by acid guanidinium thiocyanate-phenol-chloroformextractionrdquo Analytical Biochemistry vol 162 no 1 pp 156ndash1591987

[37] M C C do Espırito-Santo M V Alvarado-Mora P L S PintoF J Carrilho J R R Pinho and R C B Gryschek ldquoTwosequential PCR amplifications for detection of Schistosomamansoni in stool samples with low parasite loadrdquo Revista doInstituto de Medicina Tropical de Sao Paulo vol 54 no 5 pp245ndash248 2012

[38] M C Espırito-Santo M V Alvarado-Mora E Dias-Neto etal ldquoEvaluation of real-time PCR assay to detect Schistosomamansoni infections in a low endemic settingrdquo BMC InfectiousDiseases vol 14 no 1 article 558 2014

[39] J Hamburger T Turetski I Kapeller and R DeresiewiczldquoHighly repeated short DNA sequences in the genome ofSchistosoma mansoni recognized by a species-specific proberdquoMolecular and Biochemical Parasitology vol 44 no 1 pp 73ndash80 1991

[40] H M Santana Teles M E De Carvalho C Santos Ferreira FZacharias V R De Lima and M L Condino Fadel ldquoSchis-tosomiasis mansoni in Bananal (State of Sao Paulo Brazil) IEfficiency of diagnostic and treatment proceduresrdquo Memoriasdo Instituto Oswaldo Cruz vol 97 no 1 pp 181ndash186 2002

[41] B A de Noya C Colmenares H Lanz M A Caracciolo SLosada and O Noya ldquoSchistosomamansoni immunodiagnosisis improved by sodium metaperiodate which reduces cross-reactivity due to glycosylated epitopes of soluble egg antigenrdquoExperimental Parasitology vol 95 no 2 pp 106ndash112 2000

[42] M M L Goncalves M G M Barreto R H S Peralta et alldquoImmunoassays as an auxiliary tool for the serodiagnosis ofSchistosomamansoni infection in individuals with low intensityof egg eliminationrdquo Acta Tropica vol 100 no 1-2 pp 24ndash302006

[43] W Wang Y Li H Li et al ldquoImmunodiagnostic efficacy ofdetection of Schistosoma japonicum human infections in Chinaa meta analysisrdquo Asian Pacific Journal of Tropical Medicine vol5 no 1 pp 15ndash23 2012

[44] A W Cheever ldquoA quantitative post-mortem study of Schistoso-miasis mansoni in manrdquo American Journal of Tropical Medicineand Hygiene vol 17 no 1 pp 38ndash64 1968

[45] D Engels E Sinzinkayo S J de Vlas and B GryseelsldquoIntraspecimen fecal egg count variation in Schistosoma man-soni infectionrdquo The American Journal of Tropical Medicine andHygiene vol 57 no 5 pp 571ndash577 1997

[46] J M Ruiz ldquoCercaria species indexes of Schistosoma mansoniverified inNeves andMariana State ofMinasGeraisrdquoMemoriasdo Instituto Butantan vol 24 no 1 pp 63ndash65 1952

[47] R Correa-Oliveira L M S Dusse I R C Viana D G ColleyO S Carvalho and G Gazzinelli ldquoHuman antibody responsesagainst schistosomal antigens I Antibodies from patients withAncylostoma Ascaris lumbricoides or Schistosoma mansoniinfections react with schistosome antigensrdquo The AmericanJournal of TropicalMedicine andHygiene vol 38 no 2 pp 348ndash355 1988

[48] H Y Kanamura L C D S Dias R M Da Silva et al ldquoAcomparative epidemiologic study of specific antibodies (IgMand IgA) and parasitological findings in an endemic area of lowtransmission of Schistosoma mansonirdquo Revista do Instituto deMedicina Tropical de Sao Paulo vol 40 no 2 pp 85ndash91 1998

[49] K Kloetzel ldquoA reacao de precipitacao periovular na esquistos-somose II Correlacao aos dados clınicosrdquo Revista do Institutode Medicina Tropical de Sao Paulo vol 1 pp 129ndash137 1959

[50] B Alarcon de Noya R Ruiz S Losada et al ldquoDetectionof schistosomiasis cases in low-transmission areas based oncoprologic and serologic criteria The Venezuelan experiencerdquoActa Tropica vol 103 no 1 pp 41ndash49 2007

[51] C M Coker and J Oliver-Gonzalez ldquoExperiments on anti-schistosome circumoval precipitating antibody in micerdquo TheAmerican Journal of Tropical Medicine and Hygiene vol 7 p390 1957

[52] L B Senterfit ldquoImmobilization of the miracidia of Schistosomamansoni by immune sera II The occurrence of the antibodyduring the course of schistosomiasisrdquo American Journal ofEpidemiology vol 68 no 2 pp 148ndash155 1958

[53] J Oliver-Gonzalez P M Bauman and A S BenensonldquoImmunological aspects of infections with Schistosoma man-sonirdquo The American Journal of Tropical Medicine and Hygienevol 4 no 3 pp 443ndash452 1955

[54] D Wichmann M Panning T Quack et al ldquoDiagnosingschistosomiasis by detection of cell-free parasiteDNA inhumanplasmardquo PLoS Neglected Tropical Diseases vol 3 no 4 articlee422 2009

[55] L Zhou J Tang Y Zhao et al ldquoA highly sensitive TaqMan real-time PCR assay for early detection of Schistosoma speciesrdquoActaTropica vol 120 no 1-2 pp 88ndash94 2011

[56] R J Ten Hove J J Verweij K Vereecken K Polman LDieye and L van Lieshout ldquoMultiplex real-time PCR for thedetection and quantification of Schistosoma mansoni and Shaematobium infection in stool samples collected in northernSenegalrdquo Transactions of the Royal Society of Tropical Medicineand Hygiene vol 102 no 2 pp 179ndash185 2008

[57] D Wichmann S Poppert H Von Thien et al ldquoProspectiveEuropean-wide multicentre study on a blood based real-timePCR for the diagnosis of acute schistosomiasisrdquo BMC InfectiousDiseases vol 13 no 1 article 55 2013

[58] L Cnops E Tannich K Polman J Clerinx and M VanEsbroeck ldquoSchistosoma real-time PCR as diagnostic tool forinternational travellers and migrantsrdquo Tropical Medicine andInternational Health vol 17 no 10 pp 1208ndash1216 2012

[59] N Katz A Chaves and J Pellegrino ldquoA simple device forquantitative stool thick-smear technique in Schistosomiasismansonirdquo Revista do Instituto de Medicina Tropical de SaoPaulo vol 14 no 6 pp 397ndash400 1972

[60] WHO Expert Committee on the Control of Schistosomi-asis ldquoPrevention and control of schistosomiasis and soil-transmitted helminthiasis report of aWHO expert committeerdquoWHO Technical Report Series 912 World Health Organiza-tion Geneva Switzerland 2002 httpwhqlibdocwhointtrsWHO TRS 912pdf

[61] M J Enk A C L Lima S C Drummond V T Schall andP M Z Coelho ldquoThe effect of the number of stool sampleson the observed prevalence and the infection intensity withSchistosoma mansoni among a population in an area of lowtransmissionrdquoActa Tropica vol 108 no 2-3 pp 222ndash228 2008

[62] Y-Y Zhang J-P Luo Y-M Liu et al ldquoEvaluation of Kato-Katzexamination method in three areas with low-level endemicity


of schistosomiasis japonica in China a Bayesian modelingapproachrdquo Acta Tropica vol 112 no 1 pp 16ndash22 2009

[63] WHO World Health Organization Elimination of Schistosomi-asis from Low-Transmission Areas Report of a WHO InformalConsultation Salvador Bahia Brazil 18-19 Aug 2008 WHOGeneva Switzerland 2009

[64] Y-C Zhu ldquoImmunodiagnosis and its role in schistosomiasiscontrol in China a reviewrdquo Acta Tropica vol 96 no 2-3 pp130ndash136 2005

[65] B A de Noya R Ruiz-Guevara C Colmenares S Losadaand O Noya ldquoLow transmission areas of schistosomiasis inVenezuela consequences on the diagnosis treatment andcontrolrdquoMemorias do Instituto Oswaldo Cruz vol 101 no 1 pp29ndash35 2006

[66] H Y Kanamura L C S Dias C M Glasser et al ldquoDetection ofIgM antibodies to Schistosomamansoni gut-associated antigensfor the study of the dynamics of schistosomiasis transmission inan endemic area with low worm burdenrdquo Revista do Instituto deMedicina Tropical de Sao Paulo vol 40 no 4 pp 225ndash231 1998

[67] F Zacharias M E De Carvalho C Gargioni H M SantanaTeles C S Ferreira and V R De Lima ldquoSchistosomiasis man-soni in Bananal (State of Sao Paulo Brazil) III Seroepidemi-ological studies in the Palha districtrdquo Memorias do InstitutoOswaldo Cruz vol 97 no 1 pp 19ndash22 2002

[68] L M A Oliveira H L C Santos M M L Goncalves M GM Barreto and J M Peralta ldquoEvaluation of polymerase chainreaction as an additional tool for the diagnosis of low-intensitySchistosoma mansoni infectionrdquo Diagnostic Microbiology andInfectious Disease vol 68 no 4 pp 416ndash421 2010

[69] L Spencer B Alarcon de Noya O Noya and G MasroualdquoComparative analysis between the circumoval precipitin testand ELISA with raw antigens for the diagnosis of schistosomi-asis in Venezuelardquo GEN vol 45 no 2 pp 77ndash83 1991

[70] B A Alarcon de Noya O Noya C Balzan and I M CesarildquoNew approaches for the control and eradication of schistoso-miasis in VenezuelardquoMemorias do Instituto Oswaldo Cruz vol87 pp 227ndash231 1992

[71] T R Carneiro M C Cunha Pinheiro S M de Oliveira A Lde Paula Hanemann J A Nogueira Queiroz and F S MoraesBezerra ldquoIncreased detection of schistosomiasis withKato-Katzand SWAP- IgG-ELISA in a Northeastern Brazil low-intensitytransmission areardquo Revista da Sociedade Brasileira de MedicinaTropical vol 45 no 4 pp 510ndash513 2012

[72] L I Gomes L H D S Marques M J Enk M C de OliveiraP M Z Coelho and A Rabello ldquoDevelopment and evaluationof a sensitive PCR-ELISA system for detection of Schistosomainfection in fecesrdquo PLoS Neglected Tropical Diseases vol 4 no4 article e664 2010

[73] L A Pontes E Dias-Neto and A Rabello ldquoDetection bypolymerase chain reaction of Schistosoma mansoni DNA inhuman serum and fecesrdquo The American Journal of TropicalMedicine and Hygiene vol 66 no 2 pp 157ndash162 2002

[74] P M Z Coelho A D Jurberg A A Oliveira and N KatzldquoUse of a saline gradient for the diagnosis of schistosomiasisrdquoMemorias do Instituto Oswaldo Cruz vol 104 no 5 pp 720ndash723 2009

[75] C Fagundes Teixeira E Neuhauss R Ben J Romanzini andC Graeff-Teixeira ldquoDetection of Schistosoma mansoni eggs infeces through their interaction with paramagnetic beads in amagnetic fieldrdquo PLoS Neglected Tropical Diseases vol 1 no 2article e73 2007

[76] Z Demerdash S Mohamed M Hendawy et al ldquoMonoclonalantibody-based dipstick assay a reliable field applicable tech-nique for diagnosis of Schistosoma mansoni infection usinghuman serum and urine samplesrdquo Korean Journal of Parasitol-ogy vol 51 no 1 pp 93ndash98 2013

[77] A M Deelder N de Jonge Y E Fillie et al ldquoQuantitativedetermination of circulating antigens in human schistosomiasismansoni using an indirect hemagglutination assayrdquoThe Ameri-can Journal of Tropical Medicine and Hygiene vol 40 no 1 pp50ndash54 1989

[78] L van Lieshout U Gangaram Panday N de Jonge et alldquoImmunodiagnosis of schistosomiasis mansoni in a lowendemic area in Surinam by determination of the circulatingantigens CAA and CCArdquo Acta Tropica vol 59 no 1 pp 19ndash291995

[79] K Polman F F Stelma B Gryseels et al ldquoEpidemiologic appli-cation of circulating antigen detection in a recent Schistosomamansoni focus in Northern Senegalrdquo The American Journal ofTropical Medicine and Hygiene vol 53 no 2 pp 152ndash157 1995

[80] L Van Lieshout A M Polderman and A M DeelderldquoImmunodiagnosis of schistosomiasis by determination of thecirculating antigens CAA and CCA in particular in individualswith recent or light infectionsrdquo Acta Tropica vol 77 no 1 pp69ndash80 2000

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of


Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology


Molecular Biology International

GenomicsInternational Journal of


The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014


BioinformaticsAdvances in

Marine BiologyJournal of



Signal TransductionJournal of


BioMed Research International

Evolutionary BiologyInternational Journal of



Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014


Genetics Research International


Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational



Enzyme Research



Microbiology


1 Introduction

Schistosomiasis is a major public health problem with 200million people infected worldwide and 700 million peopleresiding in areas of infection risk [1 2]

In Brazil schistosomiasis has been reported to occur in19 states and it is estimated that approximately 6 millionpeople are infected and 25 million are at risk of contractingthe diseaseThenational positivity rate is 694 ranging from004 in Piauı State to 1188 in Pernambuco State In Rio deJaneiro State the positivity rate is 156 [3]

Brazil has areas of different prevalence rates varying fromstate to state as shown in Figure 1 [3]

Of the various known species of Schistosoma S mansonihas the widest global distribution and is the only species thatcauses schistosomiasis in Brazil [4]

Although the serious forms of schistosomiasis havebecome less prevalent thanks mainly to the implementationof mass chemotherapy the geographic expansion of schisto-somiasis continues apace with the expansion of agriculturalzones and irrigated areas [5]

The classification of the individual infection intensitycriteria for S mansoni is estimated by the quantity of eggsobserved in parasitological examination of feces using theKato-Katz (KK) technique [2] According to WHO 2002 [6]the infection classification is high parasitic load (ge400 epg)medium parasite load (100ndash399 epg) and low parasite load(lt100 epg) and areas with high medium or low endemicityshow prevalence of ge50 ge10 lt50 or lt10 respectivelyIn ALEs approximately 75 of infected individuals areasymptomatic show few symptoms and have low parasiteload which hinders diagnosis [6]

The state of Rio de Janeiro presents the lowest numberof confirmed cases and deaths due to schistosomiasis in thesoutheast region of Brazil [3 7] The city of Barra Mansa isdefined as microregion 2 of the Vale do Medio Paraıba Riode Janeiro State [8] The city of Barra Mansa is located in thesouthern part of the state of Rio de Janeiro

Barra Mansa is one of the foci of S mansoni infectionin the state of Rio de Janeiro [8] The average prevalencewas estimated to be 1 from 2001 to 2008 based on thecases reported by the Notifiable Diseases Information System(SINAN) from 2001 to 2008 [9]

The endemic foci lie within the urban perimeter Theneighborhood of Siderlandia shows the highest prevalencefollowed by the neighborhoods of Santa Clara Sao LuizCantagalo andNova Esperanca Isolated cases of infection byS mansoni have been reported in further 30 neighborhoods[9]

Detection of S mansoni eggs in feces has historicallybeen used as the reference for diagnosing schistosomiasisand Schistosoma species are identified by their characteristicmorphology showing a lateral spicule The parasitologicalmethods are highly specific inexpensive and relatively sim-ple to execute [2 10ndash12] The Kato-Katz (KK) techniqueis most commonly used for detecting S mansoni eggs inepidemiological studies allowing the quantification of eggsin fecal samples The Hoffman technique (HH) is basedon spontaneous sedimentation and it is effective because

0 250 500 1000

(km)

W E

N

S

No data

lt5

gt15

Positivity ranges ()2003ndash2012

5ndash15

Figure 1 Distribution of positivity ranges for schistosomiasis basedon the record of cases on investigated cities Brazil 2012 SourceSISPCE-SVSMS

embryonated S mansoni eggs are heavy however it is notsuitable for quantification of eggs in feces

Although these parasitological methods are inexpensiveand simple to perform they lack sensitivity especially inALEs [13ndash18] The Secretariat of Health Vigilance in Brazilhas proposed the elimination of this form of helminthiasisTherefore there is a need to define alternative laboratorydiagnostic techniques for detection of S mansoni in ALEsThus the aim of this study was to compare the efficiencyof existing parasitological immunological and moleculardiagnostic methods in areas of low prevalence of S mansoniusing the circumoval precipitin test (COPT) as a referencedue to its high sensitivity and specificity in ALEs [19 20]

2 Materials and Methods

21 Study Design Population and Sample Size S mansoniis endemic in the city of Barra Mansa Rio de Janeiro StateBrazil with an estimated prevalence of 1 [9] Data for2001ndash2008 from the Notifiable Diseases Information System(SINAN) showed that the disease is most prevalent in theneighborhoods of Siderlandia Santa Clara Sao Luiz Nova






















7

6

5

4

3

2

1

0


activ

ity in

dex
























(a) (b) (c)




















3 Results































4 Discussion
















KK-HHNegative 542 (948) 25 (44) 567 (991)






IFT-IgMNegative 508 (830) 7 (11) 515 (842)



0001 0311 0176 0446Positive 40 (70) 15 (26) 55 (96)Total 543 (949) 29 (51) 572 (1000)










KK-HH


ELISA-IgG


ELISA-IgM


IFT-IgM


qPCR-feces


qPCR-serum


































Negative COPT















Clinicalevaluation


health education

Clinicalevaluation


health education




COPT

Positive Negative






10

15

20

25

30

35

40

45


Ct








Acknowledgments


References






























































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology






















7

6

5

4

3

2

1

0


activ

ity in

dex
























(a) (b) (c)




















3 Results































4 Discussion
















KK-HHNegative 542 (948) 25 (44) 567 (991)






IFT-IgMNegative 508 (830) 7 (11) 515 (842)



0001 0311 0176 0446Positive 40 (70) 15 (26) 55 (96)Total 543 (949) 29 (51) 572 (1000)










KK-HH


ELISA-IgG


ELISA-IgM


IFT-IgM


qPCR-feces


qPCR-serum


































Negative COPT















Clinicalevaluation


health education

Clinicalevaluation


health education




COPT

Positive Negative






10

15

20

25

30

35

40

45


Ct








Acknowledgments


References






























































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology















(a) (b) (c)




















3 Results































4 Discussion
















KK-HHNegative 542 (948) 25 (44) 567 (991)






IFT-IgMNegative 508 (830) 7 (11) 515 (842)



0001 0311 0176 0446Positive 40 (70) 15 (26) 55 (96)Total 543 (949) 29 (51) 572 (1000)










KK-HH


ELISA-IgG


ELISA-IgM


IFT-IgM


qPCR-feces


qPCR-serum


































Negative COPT















Clinicalevaluation


health education

Clinicalevaluation


health education




COPT

Positive Negative






10

15

20

25

30

35

40

45


Ct








Acknowledgments


References






























































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology










3 Results































4 Discussion
















KK-HHNegative 542 (948) 25 (44) 567 (991)






IFT-IgMNegative 508 (830) 7 (11) 515 (842)



0001 0311 0176 0446Positive 40 (70) 15 (26) 55 (96)Total 543 (949) 29 (51) 572 (1000)










KK-HH


ELISA-IgG


ELISA-IgM


IFT-IgM


qPCR-feces


qPCR-serum


































Negative COPT















Clinicalevaluation


health education

Clinicalevaluation


health education




COPT

Positive Negative






10

15

20

25

30

35

40

45


Ct








Acknowledgments


References






























































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

















4 Discussion
















KK-HHNegative 542 (948) 25 (44) 567 (991)






IFT-IgMNegative 508 (830) 7 (11) 515 (842)



0001 0311 0176 0446Positive 40 (70) 15 (26) 55 (96)Total 543 (949) 29 (51) 572 (1000)










KK-HH


ELISA-IgG


ELISA-IgM


IFT-IgM


qPCR-feces


qPCR-serum


































Negative COPT















Clinicalevaluation


health education

Clinicalevaluation


health education




COPT

Positive Negative






10

15

20

25

30

35

40

45


Ct








Acknowledgments


References






























































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology











KK-HHNegative 542 (948) 25 (44) 567 (991)






IFT-IgMNegative 508 (830) 7 (11) 515 (842)



0001 0311 0176 0446Positive 40 (70) 15 (26) 55 (96)Total 543 (949) 29 (51) 572 (1000)










KK-HH


ELISA-IgG


ELISA-IgM


IFT-IgM


qPCR-feces


qPCR-serum


































Negative COPT















Clinicalevaluation


health education

Clinicalevaluation


health education




COPT

Positive Negative






10

15

20

25

30

35

40

45


Ct








Acknowledgments


References






























































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology




KK-HH


ELISA-IgG


ELISA-IgM


IFT-IgM


qPCR-feces


qPCR-serum


































Negative COPT















Clinicalevaluation


health education

Clinicalevaluation


health education




COPT

Positive Negative






10

15

20

25

30

35

40

45


Ct








Acknowledgments


References






























































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

































Negative COPT















Clinicalevaluation


health education

Clinicalevaluation


health education




COPT

Positive Negative






10

15

20

25

30

35

40

45


Ct








Acknowledgments


References






























































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology




Clinicalevaluation


health education

Clinicalevaluation


health education




COPT

Positive Negative






10

15

20

25

30

35

40

45


Ct








Acknowledgments


References






























































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology



























































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology