Detection of Schistosoma mansoni infection by TaqMan® Real-Time PCR in a hamster model

transcript

Experimental Parasitology 143 (2014) 83–89

Contents lists available at ScienceDirect

Experimental Parasitology

journal homepage: www.elsevier .com/locate /yexpr

Detection of Schistosoma mansoni infection by TaqMan�

Real-Time PCR in a hamster model

⇑ Corresponding author.E-mail address: cristinasanto@usp.br (M.C.C. Espírito-Santo).

Maria Cristina Carvalho Espírito-Santo a,⇑, Mónica Viviana Alvarado-Mora b, Pedro Luiz Silva Pinto c,Thales de Brito a,e, Lívia Botelho-Lima b, Ashley Richard Heath g, Maria Galli Amorim f,Emmanuel Dias-Neto f,h, Pedro Paulo Chieffi d,e, João Renato Rebello Pinho b, Flair José Carrilho b,Expedito José Albuquerque Luna e, Ronaldo Cesar Borges Gryschek a

a Department of Infectious and Parasitic Diseases and Laboratory of Medical Investigation 6, Faculdade de Medicina da Universidade de São Paulo, Brazilb Laboratory of Tropical Gastroenterology and Hepatology, Department of Gastroenterology, Faculdade de Medicina da Universidade de São Paulo, Brazilc Department of Enteroparasites of the Center of Parasitology and Mycology, Instituto Adolfo Lutz da Secretaria de Estado da Saúde de São Paulo, Brazild Faculdade de Ciências Médicas da Santa Casa de São Paulo, São Paulo, Brazile Instituto de Medicina Tropical de São Paulo, Universidade de São Paulo, Brazilf Laboratory of Medical Genomics, Center for International Research and Education (CIPE), Hospital AC Camargo, Brazilg Sigma Life Sciences, Woodlands, TX, USAh Laboratory of Neurociencies (LIM-27), Department and Institute of Psychiatry, Faculdade de Medicina da Universidade de São Paulo, Brazil

h i g h l i g h t s

� Detection of Schistosoma mansoniDNA by Real-Time PCR in anexperimental model.� qPCR was compared with the

Circumoval Precipitin Test and theKato-Katz technique.� First detection of eggs in feces by

Kato-Katz and qPCR feces occurred 49DPI.� S. mansoni DNA was detected in

serum samples from 14 DPI and COPTat 35 DPI.� Serum is a trustworthy source of DNA

in the pre patent infection period.

g r a p h i c a l a b s t r a c t

a r t i c l e i n f o

Article history:Received 8 January 2014Received in revised form 11 May 2014Accepted 13 May 2014Available online 22 May 2014

Keywords:S. mansoniTaqMan� Real-Time PCR systemCircumoval precipitin testKato-Katz methodHistopathology

a b s t r a c t

An experimental study in hamsters was performed to evaluate the capability for detecting Schistosomamansoni DNA in serum and fecal samples during the pre and post-egg-laying periods of infection usingTaqMan� Real-Time PCR system (qPCR), was compared with the circumoval precipitin test (COPT) andthe Kato-Katz technique, especially among individuals with low parasitic burden. Twenty-four hamsterswere infected with cercariae. Three hamsters were sacrificed per week under anesthesia, from 7 days postinfection (DPI) up to 56 DPI. A serum sample and a pool of feces were collected from each hamster. Thepresence of S. mansoni eggs in fecal samples was evaluated by Kato-Katz method and in the hamsters gut-by histopathology. Detection of S. mansoni DNA was performed using qPCR and S. mansoni antibody usingCOPT. The first detection of eggs in feces by Kato-Katz method and S. mansoni DNA in feces by qPCRoccurred 49 DPI. Nevertheless, S. mansoni DNA was detected in serum samples from 14 up to 56 DPI. COPTwas positive at 35 DPI. The results not only confirm the reliability of S. mansoni DNA detection by qPCR, butalso demonstrate that serum is a trustworthy source of DNA in the pre patent infection period.

84 M.C.C. Espírito-Santo et al. / Experimental Parasitology 143 (2014) 83–89

1. Introduction

Schistosomiasis is one of the most important parasitic diseasesin the world with a significant socioeconomic impact (King et al.,2005). It is currently estimated that 200 million people areinfected, and 700 million people live in areas at risk of infection(Chitsulo et al., 2000). Refugee movements and religious activities,as well tourism and freshwater management, are among theelements that can promote the spread of this disease (Patz et al.,2000; Ross et al., 2002).

Schistosomiasis is a neglected disease that is still present in 19Brazilian federal units, including with areas of high, medium andlow endemicity that generally compromise low-income popula-tions (Ministry of Health Brazil, 2012). In Brazil, the only speciesof medical interest is Schistosoma mansoni (Bergquist, 2002).

Whereas the syndromes associated with schistosomiasisdepend on the stage of infection, the early onset of symptomsdepends on several factors, including previous exposure to theparasite, parasitic burden and the patient’s immune response.Therefore, schistosomiasis clinical picture varies from asymptom-atic, cercarial dermatitis or acute schistosomiasis to classical syn-dromes resulting from eggs deposition in tissues (Lambertucci,2010; Gryseels, 2012).

Another aspect that should be considered is the high frequencyof asymptomatic carriers, individuals who have a role in maintain-ing the transmission of schistosomiasis in areas without adequatesanitation (Teles et al., 2002).

Classically, microscopic detection of eggs of S. mansoni in fecalsamples has been considered the gold standard for diagnosis andconfirmation of schistosomiasis in patients living in endemic coun-tries (Katz et al., 1972; WHO, 2002). Nevertheless, certain factorscontribute to decrease the sensitivity of this method: variation inthe excretion of eggs by individuals (Barreto et al., 1978; vanEtten et al., 1997); infection intensity (Ye et al., 1998); parasitismby immature forms during the pre egg-laying phase of infection(Cheever, 1968); immunity to egg products (Doenhoff et al.,1993; Karanja et al., 1997); unisexual infections (Cheever, 1968);presence of tissue injury (Ruiz-Tiben et al., 1979) and host’simmune status (Doenhoff et al., 1993; Karanja et al., 1997).

Likewise, anti-S. mansoni antibodies can be detected by variousserological methods, including immunofluorescence (IFA), indirecthemagglutination (IH), ELISA (Enzyme-Linked ImmunosorbentAssay) (Feldmeier and Büttner, 1983) and COPT (Oliver-Gonzalez,1954; Spencer et al., 1991). Thus, the inability of serological meth-ods to discriminate between acute and past infection should betaken into consideration as this limits their clinical value regardingthe possibility of a good therapeutic outcome (Rabello et al., 1997).

Thus, the aim of this study was to utilize a qPCR assay standard-ized by our group to detect S. mansoni DNA in serum and fecal sam-ples, together with assessment of the COPT and the Kato-Katztechnique during the experimental infection.

This methodology aims to improve the diagnostic sensitivity inthe pre and post-egg-laying periods and to verify whether it is pos-sible to achieve early diagnosis of the infection in the pre-egg-lay-ing period in serum samples, considering that is easier to obtainserum than fecal samples.

2. Materials and methods

2.1. Ethical aspects

This experimental study using hamsters followed the laws 6.638/79 and 9605/98, Decree 24.645/34 for Ethical Principles in AnimalExperimentation of The Brazilian College of Animal Experimentation

(COBEA), according to the Principles for Research involving Animals(Geneva, 1985).

The protocol was approved by the Ethics Committee on AnimalResearch of the Institute of Tropical Medicine (IMT) of the Univer-sity of São Paulo (USP), São Paulo, Brazil (CEP IMT-2011/096).

2.2. Experimental S. mansoni infection

The S. mansoni experimental cycle has been maintained in ham-sters and in Biomphalaria glabrata strain BH at the Laboratory ofSchistosomiasis Immunopathology (LIM-06) of the HelminthologyLaboratory of the IMT-USP.

We used 32 male hamsters aged six weeks-old, raised at theAnimal Research Facilities of the IMT-USP following conventionalsanitary standards. After physical restraint, 24 hamsters were sub-cutaneously inoculated with 150 S. mansoni cercariae, using24 � 8 mm hypodermic needles.

Following inoculation, the hamsters were placed into threelarge cages (49 � 34 � 16 cm), where they received food and waterthroughout the experiment. To assess the infection levels in the preand post-egg-laying periods, three hamsters were sacrificedweekly from 7 days post infection (week 1) up to 56 DPI (week8). Following the animal sacrifice guidelines of our institution,the hamsters were anesthetized with an intramuscular injectionof 100 mg/kg of ketamine and xylazine.

As a negative control group, 8 hamsters were not inoculatedwith S. mansoni and were followed up like the inoculated animals.One animal form the control group was sacrificed per week andwas utilized in the parasitological, histopathological, COPT andqPCR studies described below.

The sacrificed hamsters were submitted to exsanguination bypuncturing the inferior vena cava. The serum of each hamsterwas separated by centrifugation (1500�g for 15 min) and storedat �20 �C until DNA extraction and serological testing.

To detect S. mansoni DNA and perform the Kato-Katz technique,fecal samples obtained from the end portion of the bowel at thetime of sacrifice were used.

Histopathology screen was used to assess the degree of infec-tion by detection of immature forms of the parasite from thepre-egg-laying period and adult worms and eggs from the post-egg-laying period in the lung, spleen, kidney, intestines and liver.In the post-egg-laying period, control of the parasite infectionwas also performed by searching for eggs in feces using the Kato-Katz technique (Katz et al., 1972).

2.3. Preparation of histological slides

The organs were previously fixed in 10% neutral buffered for-malin, pH 7.2, embedded in paraffin, 3–4 lm sections wereobtained and stained with hematoxylin–eosin (HE). The slideswere examined under light microscopy using an Olympus CX41microscope.

2.4. The circumoval precipitin test (COPT)

This technique was used to detect antibodies against excretory/secretory antigens of miracidia. Suspensions of mature eggs iso-lated from the liver and intestines of hamsters infected with 150cercariae of S. mansoni were used, according to Dresden andPayne (1981) and modified by Pinto et al. (1995). Fifty microlitersof serum from each hamster were incubated at 37 �C for 48 h with15 lL of suspension containing 200 mature eggs. Reading criteriawas based on Spencer et al. (1991), Noya et al. (2002) and Noyaet al. (2006), who considered as positive reaction when therewas the presence of periovular precipitates in at least 9% of theeggs.

M.C.C. Espírito-Santo et al. / Experimental Parasitology 143 (2014) 83–89 85

2.5. Standardization of S. mansoni DNA detection by qPCR

From the laboratory cycle, S. mansoni eggs were extracted andcleaned according to Dresden and Payne (1981) and Pinto et al.(1995), as previously described. This solution was resuspended ina 0.9% saline solution and stored at �20 �C until use.

As a positive control for PCR in serum, a suspension of 200 eggs/mL in 0.9% saline was submitted to DNA extraction. For PCR posi-tive control in feces, we used DNA extracted from 500 mg offeces of uninfected hamsters label mixed with approximately200 eggs/mL.

2.6. DNA extraction from serum

DNA extraction from the serum of hamsters and the 0.9% salinesolution containing 200 eggs/mL was performed using the guanidineisothiocyanate-phenol-chloroform (GT) method (Chomczynski andSacchi, 1987; Espírito-Santo et al., 2012). DNA was stored at�20 �C after extraction.

2.7. DNA from fecal samples

DNA extraction from the feces of hamsters was performed intwo phases: in the first phase, after resuspending approximately500 mg of stool in 1000 lL 0.1 M PBS, five glass beads were added.Subsequently, this mixture was homogenized for 5 min using avortex mixer, followed by centrifugation for 8 min at 13,200 rpmat 4 �C. An aliquot of 400 lL of the supernatant was mixed with100 lL of ROSE (Rapid One-Step Extraction) solution: 10 mMTris-hydroxymethyl amino methane HCl, pH 8, 300 mM (EDTA),pH 8.0, 1% Sarkosyl (sodium lauryl sarcosinate); 1% PVPP (Polyvi-nylpolypyrrolidone) (Steiner et al., 1995). Then, 30 lL of ProteinaseK (Life Technologies, Carlsbad, California, USA) was added andhomogenized using a vortex mixer. The sample was incubatedfor 120 min at 65 �C. In the second phase, the DNA present in thissolution was extracted and stored as described for serum samples.

2.8. Purification of DNA extracted from fecal and serum

DNA extracted from serum and feces, collected at distinct timespost infection, were purified using the InstaGene Matrix in accor-dance with the manufacturer’s instructions. Once purified,extracted DNA was stored at �20 �C until use.

2.9. Amplification of DNA from serum and fecal samples

2.9.1. Primers and probesDNA samples were amplified and detected using a set of prim-

ers and probes complementary to a 121 bp tandem repeatsequence of S. mansoni strain SM 1–7 (GenBank accession numberM61098) described by Hamburger et al. (1991). Primer sequenceswere: forward F2: 50-CCG ACC AAC CGT TCT ATG A-30; reverse R2:50-CAC GC TCT CGC AAA TAA TCT AAA-30 (Pontes et al., 2002);probe PO2: 50-6[FAM] TCG TTG TAT CTC CGA AAC CAC TGG ACG[(BHQ1])-30, all synthesized by Sigma Life Sciences (Woodlands,Texas, USA).

All the samples were evaluated using TaqMan� Reagents Exog-enous Internal Positive Control (IPC) (Life Technologies), in accor-dance with the manufacturer’s instructions to check for thepresence of Taq DNA polymerase inhibitors.

2.10. TaqMan� Real-Time conditions of serum and fecal samples

TaqMan� Real-Time PCR was performed in a final volume of20 lL containing: 10 lL TaqMan� Universal PCR Master Mix 2 X;20 pmol of primers F2 and R2, 5 pmol of the PO2 probe and 2 lL

of purified DNA. For each sample, another reaction was performedin parallel using the TaqMan� Reagents Exogenous Internal Posi-tive Control (IPC) a in the final volume of 21 lL, containing:10 lL of TaqMan� Universal PCR Master Mix 2X, 5 lL 10X Exoge-nous IPC mix, 1 lL 50X Exo IPC and 5 lL of purified DNA samples.For each batch of reactions, two other controls were used: NAC (NoAmplification Control) and NTC (No Template Control Target). PCRwas performed in an Applied Biosystems 7300 Real-Time PCR Sys-tem� (Life Technologies) using the following cycling conditions:50 �C for 2 min; 95 �C for 10 min; and 40 cycles at 95 �C for 15 sand 60 �C for 1 min.

To detect S. mansoni in the reaction, the Applied Biosystems7300 Real-Time PCR System� standard cycles were used: 50 �Cfor 2 min; 95 �C for 10 min; 40 cycles at 95 �C for 15 s, and 60 �Cfor 1 min.

Furthermore, the possibility of contamination was minimizedby the physical separation of the rooms where DNA extractionand DNA amplification were performed, by performing all theexperiments inside a lamina flow cabinet, by frequent use of UVirradiation and by using only disposable sterile laboratory equip-ments and pipette tips with filters.

2.11. Sensitivity and specificity of qPCR

To assess the specificity of the PCR assay here standardized, weused DNA extracted from other parasites (Ascaris lumbricoides andStrongyloides stercoralis). Similarly, the specificity of the primersand probes was also investigated using BLAST against public dat-abases in the NCBI (www.ncbi.nlm.nih.gov). The results showedno predicted amplification from other parasite species, includingother Schistosome species (data not shown).

Analytical sensitivity was assessed by DNA detection using PCRstandardized from a serial dilutions assay, starting with 200 eggs ofS. mansoni in 0.9% saline. This sample was diluted 5 times: puresample (200 eggs); 1:10 (20 eggs); 1:100 (2 eggs); 1:1000 (0.2eggs); 1:(10,000 (0.02 eggs); and 1:100,000 (0.002 eggs).

3. Results

3.1. Analysis of histological slides

Liver sections of the three hamsters sacrificed in the initialphase of infection (day 14) revealed mild chronic hepatitis, charac-terized by discrete fibrous enlargement of portal spaces with thepresence of lymphocytic infiltrate, predominant lymphocyte andpreserved limiting plate. At 21 and 28 DPI, chronic hepatitisbecame moderate with marked expansion of portal infiltrate.These changes were present in all three hamsters. Together withhepatitis, the presence of schistosomula pairs was observed at 28DPI in two hamsters. At 35 DPI, chronic hepatitis was intense, withmarked portal infiltrate and aggression limiting plate of the hepaticlobule. The presence of adult worms was seen in one hamster, andin others, worms and eggs of S. mansoni were observed. An intenseinflammatory process was maintained up to 56 DPI.

Sections of small intestine revealed mild to moderate inflam-matory infiltrate in the corium at 14, 21 and 28 DPI in all threeinfected hamsters. S. mansoni eggs were present in the small intes-tine, intravascularly, in two hamsters at 28 DPI. At 35, 42 and from49 to 56 DPI, lymphocytic inflammation became more intense andthe appearance of epithelioid granulomas was observed around theeggs, which were usually viable. We also observed the presence ofworms in two hamsters at 42 DPI.

Sections of large intestine revealed mild inflammatory infiltratefrom 14 to 35 DPI. The presence of S. mansoni eggs in the wall of thelarge intestine was observed only after 49 DPI, when the

lymphomononuclear inflammatory process became more intensewith the appearance of epithelioid granulomas around the eggs.

Lungs showed foci of interstitial pneumonitis with edema andlymphocytic infiltration 14 DPI. By 35 DPI, the occasional presenceof schistosomula with S. mansoni mated in smaller branches of thepulmonary artery and eggs of the parasite in the lung parenchymawere observed. Pneumonitis increased by 56 DPI, following by thepresence of parasite eggs in the lung parenchyma.

Spleen slides revealed reactivity of the white pulp, whichincreased from 21 to 56 DPI in all three infected hamsters.

Kidneys showed a mesangioproliferative glomerulonephritis at28 (two hamsters) and 35 (two hamsters) DPI. However, only thepresence of a mild glomerulitis was observed in the remainingexperimental animals.

Normal controls revealed no histopathological abnormalities.

3.2. The Kato-Katz method and circumoval precipitin test (COPT)

Parasitological examination of the pool of fecal samples waspositive from 49 DPI onward.

The COPT was positive from 35 DPI and remained positive insubsequent periods (Table 1).

3.3. qPCR

3.3.1. Sensitivity and specificity of qPCRThe results of the sensitivity tests showed that S. mansoni DNA

was detected up to the 5th dilution (1/10,000), corresponding toapproximately 0.02 eggs (Ct 35.83) of S. mansoni, R2 = 0.99972.No amplification was observed for the negative controls (no DNAfrom other parasites), confirming the previous BLAST analysisresults, showing no significant matches.

3.4. Detection of DNA in serum and stool samples from hamsters

The first positive amplification signal in the serum was obtainedat 14 DPI for one hamster Cycle Threshold (Ct 37.37). At the nexttime point (21 DPI), the serum of all three hamsters were positive,with Cts ranging from 32.4 to 38.7. All the samples remained posi-tive up to 56 DPI (Fig. 1).

For fecal samples, the first positive PCR results were obtainedonly 49 days after infection for the three analyzed hamsters. PCRresults remained positive in two animals after 56 DPI (Fig. 2).

4. Discussion

In this study, we compared the results of qPCR of S. mansonifrom fecal and serum samples with those of the COPT and theKato-Katz method, in order to evaluate these approaches as diag-nostic tools for early diagnosis, including the prepatent periodand when the parasites have not reached sexual maturity and eggsare not available for disease diagnosis.

Table 1Weekly distribution of positivity in circumoval precipitin test (COPT) and the Kato-Katz stool examination technique, post infection.

Weekly samplecollection

Number ofhamsters

PositiveCOPT#

Kato-Katz pool of fecalsamples

1 3 0 02 3 0 03 3 0 04 3 0 05 3 1 06 3 2 07 3 3 Positive8 3 3 Positive

A parallel histopathological study of lung, liver, intestine, kid-ney and spleen sections from the hamsters was conducted in orderto determine associations between tissue changes identified dur-ing experimental infection and the results obtained from the labo-ratory techniques used.

Pontes et al. (2002) first reported the high sensitivity of conven-tional PCR to detect DNA in the serum of two patients with activeintestinal schistosomiasis, using a highly repetitive nucleotidesequence of S. mansoni as a target. The possibility of using qPCRto diagnose this parasite was later confirmed by Sadek et al. (2008).

More recently, Wichmann et al. (2009) used qPCR to amplify thesame 121-bp tandem repeat sequence (GenBank M61098) used byPontes et al. (2002), and detected parasite DNA in plasma collectedfrom patients with acute infection and active disease followingchemotherapy.

In an experimental study in mice, Hussein et al. (2012) detectedfree circulating S. mansoni DNA after day 3 post infection, usingconventional PCR. In this work, free circulating S. mansoni DNAwas detected in serum samples in week 2 following infection.Detection remained positive in the serum of all infected hamstersup to end of the experimental period (week 8).

Unlike the study by Hussein et al. (2012), our experimentalmodel was developed in hamsters. Cheever et al. (2002) reportedthat the murine model better reflects the human disease. Notwith-standing this fact, other aspects should be considered, including:(i) the use of hamsters in our study, since these animals have a lar-ger plasma volume compared with mice and this could lead tomore diluted parasite DNA; and (ii) the strains of parasites usedin the two studies: BH strain by us and Egyptian strain by Hussein,with possible difference in the number of tandem repeats in thetarget DNA (Hamburger et al., 1991). Nevertheless, despite thesemethodological differences, both studies verified that the earlydiagnosis of S. mansoni infection, within the prepatent period, ispossible in animal models. The early detection of parasite DNA intwo distinct animal models of schistosomiasis suggests that pre-patent detection of parasite DNA should also be possible in humanpatients.

Regarding the histopathological changes observed in this study,we verified that at least one organ of the three hamsters sacrificedper week presented alterations.

The presence of young forms of S. mansoni was identified in sec-tions of hamster lung tissue in week 5 and sections of liver tissue inweeks 4, 5 and 6. Eggs of S. mansoni were initially observed in sec-tions of the small intestine from week 4 onward, when they wereobserved frequently; from week 5, especially in sections of livertissue and in of small and small intestine tissues up to week 8.These alterations were accompanied by granulomatous inflamma-tion, which ranged in intensity from moderate to severe.

These findings coincide with the positive results in triplicatesamples of serum by PCR, except for week 5, when DNA amplifica-tion of S. mansoni serum was positive in duplicate.

These histopathological changes resemble the hypothesesraised by Zhou et al. (2011) in their experimental study involvingmice infected with S. japonicum, in which PCR was used to detectcirculating DNA of this species in serum samples.

According to Zhou et al. (2011), circulating DNA fragments of S.japonicum are produced by the degradation of schistosomula and/or eggs of this parasite due to the intense inflammatory reactionthat takes place in an attempt to arrest the infection. The authorsalso suggested that by week 4, the free circulating DNA detectedby qPCR is the result of fragments of schistosomula, young adultworms and eggs of S. japonicum. After week 4, detection of circulat-ing DNA fragments primarily results from degraded egg fragments.

Similar to our data, these authors reported a correlationbetween the detection of free circulating DNA and histopatholo-gical changes, observing an intense inflammatory reaction, the

Fig. 1. Distribution of serum qPCR positivity expressed in Cycle Threshold (Ct) values in the different post infection days.

Fig. 2. Distribution of feces qPCR positivity expressed in Cycle Threshold (Ct) values in the different post infection days.

presence of granulomas and inflammatory cells, when, due to theinstallation of fibrosis and portal hypertension (Warren et al.,1975; Blanchard, 2004), circulating DNA fragments of S. japonicumbegin to decline and their detection in serum by PCR reaction alsodecreases.

Cheever et al. (2002) reported a similar chronology of histopa-thological changes, mainly in liver sections in an experimentalstudy involving mice infected with cercariae of S. mansoni, whichalso supports the findings of this study.

Regarding fecal samples, qPCR was positive in weeks 7 and 8post infection, coinciding with the Kato-Katz technique. Thus, thePCR assay in stool samples was not sensitive enough to detectthe presence of DNA before the parasitological method, even withthe onset of the egg-laying period around week 4 post infection.This result coincides with the findings reported by Cheever et al.

(2002) in their study involving mice, which showed that adultworms begin laying eggs at around week 5, but penetration ofthe eggs through the intestinal wall may take about two weeks.Variations in the distribution and elimination of eggs in the fecesshould be taken into account when comparing the PCR results withthe delayed positive result in stool samples (Engels et al., 1996; Yeet al., 1998; Berhe et al., 2004). In addition, the possibility that themass of fecal samples examined may not have contained eggs or S.mansoni DNA fragments should be also considered (Wichmannet al., 2009).

Regarding the comparison of diagnostic methods, in this study,the serological technique used was COPT (Spencer et al., 1991),which was positive in week 5 post infection in one hamster andremained positive in two or three hamsters up to the end of thestudy. Therefore, this serological reaction was sensitive enough

to detect early infection with S. mansoni before the stool test,although serum PCR was positive after 4 weeks post infection.

COPT was positive one week after the onset of egg-laying in thesmall intestine. A study by Long et al. (1980), involving miceinfected with S. japonicum, showed the presence of vacuolatedperioval precipitate, non septate, similar to the morphologyobserved in humans with recent infection, ten weeks postinfection.According to Noya et al. (2002), COPT positivity has a direct corre-lation with egg-laying, confirming the infection status of the patentperiod.

Noya et al. (2006) reported high sensitivity and specificity forthe COPT, suggesting it could be used as the gold standard for sero-logical infections with low parasitic burden. Thus, despite beinglabor intensive, it may represent an interesting option for the earlydetection of infection by S. mansoni.

Wichmann et al. (2009) verified the early detection of DNA inplasma of patients with active disease following chemotherapy,compared with serology by EIA (Indirect Solid-Phase EnzymeImmunoassay). Hussein et al. (2012) demonstrated that the indi-rect hemagglutination assay (IHA) failed to detect infection withS. mansoni until week 8 post infection compared with conventionalPCR on serum samples.

Recent studies reported the finding of Schistosoma haematobium(Ibironke et al., 2011, 2012) but also S. mansoni DNA (Lodh et al.,2013) in the urine of infected patients.

In this study, we used the TaqMan� Real-Time system, utilizingsame primers sequences used by Wichmann et al. (2013), but anovel probe was idealized. This method was previously reportedto show higher sensitivity in serum samples of individuals withacute S. mansoni infection compared with parasitological andimmunological techniques.

Even though satisfactory results were obtained for the earlydiagnosis of S. mansoni infection using qPCR on serum and/or theCOPT, given the large difference in plasma volume between ham-sters and humans, this method should be applied in a larger num-ber of samples, particularly from low endemicity areas. Furtherapplication of these methods in other experimental settings andon human samples is required to precisely determine the validityof our findings for human diagnosis (Wichmann et al., 2009;Hussein et al., 2012).

Under the experimental conditions described, our results indi-cate that qPCR using serum-derived DNA was the first test todetect the presence of infection by S. mansoni, followed by COPT,PCR in feces and finally the parasitological Kato-Katz method.

Results from our group and others indicate the possibility ofusing serum for early diagnosis of schistosomiasis, possibly thusfilling the diagnostic gap that makes early treatment difficult toestablish what may have an impact in the treatment prognosisfor infected individuals. Furthermore, it should take into consider-ation that patients are less resistant to the use of serum samples toperform diagnostic tests rather than using stool samples(Wichmann et al., 2009).

This study corroborates the results obtained by Wichmann et al.(2013) and reinforces the use of Kato-Katz technique, COPT andPCR separately and/or in combination to diagnose acute schistoso-miasis in infections in the prepatent period, both in clinical andexperimental studies.

Acknowledgments

This study was supported by the São Paulo Research Foundationunder processes 07/53457-7, 08/50461-6 and 10/52615-0. Wewould like to thank Dra. Luciana Inácia Gomes and Dra. AnaRabello, from the Laboratório de Pesquisas Clínicas (Laboratory ofClinical Research) of the Instituto de Pesquisas René Rachou – Fio-cruz, Belo Horizonte, Brazil, for their assistance during the

standardization process. Finally, we would like to thank Miss MariaCristina Conceição de Mello from LIM-06-FMUSP for her excellenttechnical assistance concerning the S. mansoni biological cycle.

References

Barreto, M.L., Silva, J.T., Mott, K.E., Lehman Jr., J.S., 1978. Stability of faecal eggexcretion in Schistosoma mansoni infection. Trans. R. Soc. Trop. Med. Hyg. 72,181–187.

Bergquist, N.R., 2002. Schistosomiasis: from risk assessment to control. TrendsParasitol. 18, 309–314.

Berhe, N., Medhin, G., Erko, B., Smith, T., Gedamu, S., Bereded, D., Moore, R., Habte,E., Redda, A., Gebre-Michael, T., Gundersen, S.G., 2004. Variations in helminthfaecal egg counts in Kato-Katz thick smears and their implications in assessinginfection status with Schistosoma mansoni. Acta Trop. 92, 205–212.

Blanchard, T.J., 2004. Schistosomiasis. Travel Med. Infect. Dis. 2, 5–11.Cheever, A.W., 1968. A quantitative post-mortem study of Schistosomiasis mansoni

in man. Am. J. Trop. Med. Hyg. 17, 38–64.Cheever, A.W., Lenzi, J.A., Lenzi, H.L., Andrade, Z.A., 2002. Experimental models of

Schistosoma mansoni infection. Mem. Inst. Oswaldo Cruz 97, 917–940.Chitsulo, L., Engels, D., Montresor, A., Savioli, L., 2000. The global status of

chistosomiasis and its control. Acta Trop. 77, 41–51.Chomczynski, P., Sacchi, N., 1987. Single-step method of RNA isolation by acid

guanidinium thiocyanate-phenol-chloroform extraction. Anal. Biochem. 162,156–159.

Doenhoff, M.J., Butterworth, A.E., Hayes, R.J., Sturrock, R.F., Ouma, J.H., Koech, D.,Prentice, M., Bain, J., 1993. Seroepidemiology and serodiagnosis ofschistosomiasis in Kenya using crude and purified egg antigens of Schistosomamansoni in ELISA. Trans. R. Soc. Trop. Med. Hyg. 87, 42–48.

Dresden, M.H., Payne, D.C., 1981. A sieving method for the collection of schistosomeeggs from mouse intestines. J. Parasitol. 3, 450–452.

Engels, D., Sinzinkayo, E., Gryseels, B., 1996. Day-to-day egg count fluctuation inSchistosoma mansoni infection and its operational implications. Am. J. Trop.Med. Hyg. 54, 319–324.

Espírito-Santo, M.C.C., Alvarado-Mora, M.V., Pinto, P.L., Carrilho, F.J., Pinho, J.R.,Gryschek, R.C.B., 2012. Two sequential PCR amplifications for detection ofSchistosoma mansoni in stool samples with low parasite load. Rev. Inst. Med.Trop. Sao Paulo 54, 245–248.

Feldmeier, H., Büttner, D.W., 1983. Immunodiagnosis of Schistosomiasishaematobium and Schistosomiasis mansoni in man. Application of crudeextracts from adult worms and cercariae in the IHA and the ELISA. Zentralbl.Bakteriol. Mikrobiol. Hyg. A 255, 413–421.

Gryseels, B., 2012. Schistosomiasis. Infect. Dis. Clin. North Am. 26, 383–397.Hamburger, J., Turetski, T., Kapeller, I., Deresiewicz, R., 1991. Highly repeated short

DNA sequences in the genome of Schistosoma mansoni recognized by a species-specific probe. Mol. Biochem. Parasitol. 44, 73–80.

Hussein, H.M., El-Tonsy, M.M., Tawfik, R.A., Ahmed, S.A., 2012. Experimental studyfor early diagnosis of prepatent Schistosomiasis mansoni by detection of freecirculating DNA in serum. Parasitol. Res. 111, 475–478.

Ibironke, O.A., Phillips, A.E., Garba, A., Lamine, S.M., Shiff, C., 2011. Diagnosis ofSchistosoma haematobium by detection of specific DNA fragments from filteredurine samples. Am. J. Trop. Med. Hyg. 84, 998–1001.

Ibironke, O., Koukounari, A., Asaolu, S., Moustaki, I., Shiff, C., 2012. Validation of anew test for Schistosoma haematobium based on detection of Dra1 DNAfragments in urine: evaluation through latent class analysis. PLoS Negl. Trop.Dis. 6, e1464.

Karanja, D.M., Colley, D.G., Nahlen, B.L., Ouma, J.H., Secor, W.E., 1997. Studies onschistosomiasis in western Kenya: I. Evidence for immune-facilitated excretionof schistosome eggs from patients with Schistosoma mansoni and humanimmunodeficiency virus coinfections. Am. J. Trop. Med. Hyg. 56, 515–521.

Katz, N., Chaves, A., Pellegrino, J., 1972. A simple device for quantitative stool thicksmear technique in Schistosomiasis mansoni. Rev. Inst. Med. Trop. Sao Paulo 14,397–400.

King, C.H., Dickman, K., Tisch, D.J., 2005. Reassessment of the cost of chronichelmintic infection: a meta-analysis of disability-related outcomes in endemicschistosomiasis. Lancet 365, 1561–1569.

Lambertucci, J.R., 2010. Acute Schistosomiasis mansoni: revisited and reconsidered.Mem. Inst. Oswaldo Cruz 105, 422–435.

Lodh, N., Mwansa, J.C., Mutengo, M.M., Shiff, C.J., 2013. Diagnosis of Schistosomamansoni without the stool: comparison of three diagnostic tests to detectSchistosoma [corrected] mansoni infection from filtered urine in Zambia. Am. J.Trop. Med. Hyg. 89, 46–50.

Long, G.W., Pelley, R.P., Blas, B.L., Yogore Jr., M.G., Lewert, R.M., 1980. Analysis of theimmunoglobulins responsible for the circumoval precipitation reaction. Am. J.Trop. Med. Hyg. 29, 1241–1245.

Ministry of Health Brazil, 2012. Surveillance Secretariat of the Health InformationSystem of the Schistosomiasis Control Program. Information System Diseases inSaúde/2012. Confirmed cases of schistosomiasis, Brazil, Major Regions andfederal units, from 1995 to 2011.

Noya, B.A., Guevara, R.R., Colmenares, C., Losada, S., Noya, O., 2006. Lowtransmission areas of schistosomiasis in Venezuela: consequences on thediagnosis, treatment, and control. Mem. Inst. Oswaldo Cruz 101, 29–35.

Noya, O.B., de Noya, A., Losada, S., Colmenares, C., Guzmán, C., Lorenzo, M.A.,Bermúdez, H., 2002. Laboratory diagnosis of schistosomiasis in areas of low

transmission: a review of a line of research. Mem. Inst. Oswaldo Cruz 97, 167–169.

Oliver-Gonzalez, J., 1954. Anti-egg precipitins in the serum of humans infected withSchistosoma mansoni. J. Infect. Dis. 95, 86–91.

Patz, J.A., Graczyk, T.K., Geller, N., Vittor, A.Y., 2000. Effects of environmental changeon emerging parasitic diseases. Int. J. Parasitol. 30, 1395–1405.

Pinto, P.L.S., Floriano, L.D., Ferreira, S.C., Suto, L.M., Vellosa, S.A.G., 1995. Purificaçãode ovos de Schistosoma mansoni a partir de vísceras de hamsters (Crycetusauratus) experimentalmente infectados. Rev. Patol. Trop. 23, 264.

Pontes, L.A., Dias-Neto, E., Rabello, A., 2002. Detection by polymerase chain reactionof Schistosoma mansoni DNA in human serum and feces. Am. J. Trop. Med. Hyg.66, 157–162.

Rabello, A.L., Garcia, M.M., Pinto da Silva, R.A., Rocha, R.S., Katz, N., 1997. Humoralimmune responses in patients with acute Schistosoma mansoni infection whowere followed up for two years after treatment. Clin. Infect. Dis. 24, 304–308.

Ross, A.G., Bartley, P.B., Sleigh, A.C., Olds, G.R., Li, Y., Williams, G.M., McManus, D.P.,2002. Schistosomiasis. N. Engl. J. Med. 346, 1212–1220.

Ruiz-Tiben, E., Hillyer, G.V., Knight, W.B., Gómez de Rios, I., Woodall, J.P., 1979.Intensity of infection with Schistosoma mansoni: its relationship to thesensitivity and specificity of serologic tests. Am. J. Trop. Med. Hyg. 28, 230–236.

Sadek, S.M., El Missiry, A.M., El-Tonsy, M.M., Hussein, H.M., El Asmar, M.F., Ahmed,S.A., 2008. Detection of Schistosoma mansoni DNA in human blood as adiagnostic method for Schistosomiasis. Parasitologists United J. 1, 31–36.

Spencer, L., Alarcón de Noya, B., Noya, O., Masroua, G., 1991. Comparative analysisbetween the circumoval precipitin test and ELISA with raw antigens for thediagnosis of schistosomiasis in Venezuela. G E N 45, 77–83, Spanish.

Steiner, J.J., Poklemba, C.J., Fjellstrom, R.G., Elliott, L.F., 1995. A rapid one-tubegenomic DNA extraction process for PCR and RAPD analyses. Nucleic Acids Res.23, 2569–2570.

Teles, H.M., de Carvalho, M.E., Santos Ferreira, C., Zacharias, F., de Lima, V.R., Fadel,M.L., 2002. Schistosomiasis mansoni in Bananal (State of São Paulo, Brazil): I.Efficiency of diagnostic and treatment procedures. Mem. Inst. Oswaldo Cruz 97,181–186.

van Etten, L., van Lieshout, L., Mansour, M.M., Deelder, A.M., 1997. A reagent stripantigen capture assay for the assessment of cure of schistosomiasis patients.Trans. R. Soc. Trop. Med. Hyg. 91, 154–155.

Warren, K.S., Boros, D.L., Hang, L.M., Mahmoud, A.A., 1975. The Schistosomajaponicum egg granuloma. Am. J. Pathol. 80, 279–294.

Wichmann, D., Panning, M., Quack, T., Kramme, S., Burchard, G.D., Grevelding, C.,Drosten, C., 2009. Diagnosing schistosomiasis by detection of cell-free parasiteDNA in human plasma. PLoS Negl. Trop. Dis. 3, e422.

Wichmann, D., Poppert, S., Thien, H.V., Clerinx, J., Dieckmann, S., Jensenius, M.,Philippe Parola, G.D., Richter, J., Schunk, M., Stich, A., Zanger, P., Burchard, G.D.,Egbert Tannich, E., 2013. Prospective European-wide multicentre study on ablood based real-time PCR for the diagnosis of acute schistosomiasis. BMCInfect. Dis. 13, 55.

World Health Organization, 2002. TDR Reference Group on Schistosomiasis.Schistosomiasis: Strategic Direction for Research. World Health Organization,Geneva, Switzerland. Available from: <http://www.who.int/tdr/diseases/schisto/direction.htm>.

Ye, X.P., Donnelly, C.A., Anderson, R.M., Fu, Y.L., Agnew, A., 1998. The distribution ofSchistosoma japonicum eggs in faeces and the effect of stirring faecal specimens.Ann. Trop. Med. Parasitol. 92, 181–185.

Zhou, L., Tang, J., Zhao, Y., Gong, R., Lu, X., Gong, L., Wang, Y., 2011. A highly sensitiveTaqMan real-time PCR assay for early detection of Schistosoma species. ActaTrop. 120, 88–94.

Detection of Schistosoma mansoni infection by TaqMan® Real-Time PCR in a hamster model

Documents