a cross-sectional study of entamoeba histolytica/dispar...

Research ArticleA Cross-Sectional Study of Entamoebahistolyticadisparmoshkovskii Complex in SalvadorBahia Brazil

Neci M Soares 1 Helen C Azevedo1 Flaacutevia T F Pacheco1 Joelma N de Souza1

Rodrigo P Del-Rei2 Maacutercia C A Teixeira 1 and Fred L N Santos 3

1Pharmacy College Federal University of Bahia UFBA Salvador-BA 40170-115 Brazil2Faculty of Technology and Sciences of Bahia FATEC-BA Salvador-BA 40280-901 Brazil3Advanced Public Health Laboratory Goncalo Moniz Institute FIOCRUZ-BA Salvador-BA 40296-710 Brazil

Correspondence should be addressed to Fred L N Santos fredsantosbahiafiocruzbr

Received 11 April 2019 Revised 2 July 2019 Accepted 14 July 2019 Published 24 July 2019

Academic Editor Maria Carolina Touz

Copyright copy 2019 Neci M Soares et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Epidemiological studies on species-specific Entamoeba infections are scarce due to the morphological similarity of pathogenicEntamoeba histolytica and nonpathogenic E dispar and E moshkovskii The diagnosis of E histolytica is frequently based oncoproantigen (E histolytica-GalGalNAc lectin specific) detection by immunoassays However specific E histolytica-lectin is notexpressed in cysts which are eliminated by asymptomatic individuals leading to false-negative results and an underestimation ofamebiasis prevalenceMolecular techniques based on the amplification of parasiteDNAhave been shown to be a highly sensitive andspecificmethod that allows the detection of differentEntamoeba speciesThis study aimed to assess the frequency of the species fromE histolyticadisparmoshkovskii complex by molecular and immunological techniques in individuals attended at a public healthsystem in Salvador-Bahia Brazil A cross-sectional study involving 55218 individuals was carried out The diagnosis was basedon microscopy revealing E histolyticadisparmoshkovskii complex The species differentiation was performed by E histolytica-specific antigen serological evaluation and by molecular technique The overall prevalence of E histolyticadisparmoshkovskiicomplex determined by microscopy was approximately 049 (27355218) E histolytica-specific antigen detection and molecularcharacterization returned 100 negativity for E histolytica However serological evaluation returned an 89 positivity (890) Inthe stool samples analysed by PCR it was not possible to identify E histolytica and E moshkovskii although circulating IgG anti-Ehistolytica has been detected

1 Introduction

Entamoeba histolytica is a common pathogenic protozoanwidely distributed throughout the world It is responsiblefor amoebic dysentery and amoebic liver abscess resultingin human suffering and death Amebiasis is significantlyassociated with food and drinking water supplies contami-nated with human faeces and affects approximately 10 ofthe worldrsquos population with 50000-100000 deaths reportedannually making this a significant cause of death due toprotozoan parasites [1] The E histolytica prevalence isoverestimated due to its epidemiological overlap with othermorphologically identical species ie Entamoeba dispar

and Entamoeba moshkovskii currently composing the EhistolyticaE disparE moshkovskii complex [2ndash5] Besidesthe cysts of Entamoeba hartmanni another nonpathogenicamoeba even smaller in size (gt 10 120583m) can be confused withE histolytica cysts in the parasitological examination

The prevalence of each species from this complex is notwell characterized in many geographic regions It is well-established that only E histolytica leads to invasive diseasein humans Although some reports suggest a potential roleof both E dispar and E moshkovskii in provoking diseasein humans they are still considered to be nonpathogenicand free-living amoebas respectively [6ndash10] Neverthelessthe differentiation of all Entamoeba species is substantial

HindawiBioMed Research InternationalVolume 2019 Article ID 7523670 7 pageshttpsdoiorg10115520197523670

2 BioMed Research International

for preventing unnecessary and indiscriminate treatmentwith anti-amoebic chemotherapy which could lead to drugresistance [11] The World Health Organization advises thatcases determined to have E histolytica should be treatedwhether or not clinical symptoms are present [12] Addi-tionally control measures could be more efficiently appliedin geographical areas with well-known epidemiological set-tings

Regarding the low viability of trophozoites in diarrhealspecimens and irregular faecal cyst excretion in asymp-tomatic hosts the use of different diagnostic methods isrequired to increase the sensitivity of parasite identificationin faecal samples Immunoassays for coproantigen detectionof specific E histolytica-GalGalNAc lectin have been usedas alternative methods for the diagnosis of amebiasis [13]Molecular techniques based on the amplification of parasiteDNA have been shown to be a highly sensitive and specificmethod that allows the detection of different Entamoebaspecies [14] However a negative result does not rule outthe presence of the parasite due to interference from PCRinhibitors present in faeces that may hamper DNA amplifi-cation

In Brazil due to regional differences in sanitation andsocioeconomic conditions E histolyticadisparmoshkovskiicomplex distribution is irregular with 25-11 in the Southand Southeast 19 in the North and the Amazon region andapproximately 10 in the Northeast and Central West [15]In the city of Salvador the capital of the Brazilian state ofBahia the presence of the complexwas shown in some studiesconducted by our group with a prevalence of 5 and 32 inindividuals attended at public [16] and the private health sys-tem respectively Moreover 15 (2621788) of positive stoolsamples for E histolyticaE dispar complex was analysed byPCR and no DNA amplified for E histolytica DNA amplifiedfor E dispar was observed in 866 of samples (227262) [17]However in a previous study also conducted by our group46 (7153) of hospitalized diarrheal children from Salvadortested positive for specific lectin of E histolytica-GalGalNAc[18]

These findings further support evidence that E histolyticamay be infecting humans in Salvador Given the importanceof the actual prevalence of E histolytica E dispar andE moshkovskii infections this study aimed to assess thefrequency of each species by molecular and immunologicaltechniques in individuals attended at the public health systemin Salvador-Bahia Brazil

2 Materials and Methods

21 Ethical Considerations Approval was granted by theInstitutional Review Board (IRB) for Human Research atthe Goncalo Moniz Institute Oswaldo Cruz Foundation(FIOCRUZ) Salvador Bahia-Brazil registration number1002006 All procedures were conducted in strict adherenceto the principles laid out in the Declaration of HelsinkiInformed consentwas obtained fromparticipantswho agreedto participate of the study Samples were anonymously codedto protect each participantrsquos identity

22 Study Design and Population A cross-sectional studywas performed from February 2010 to June 2014 involving55218 individuals who attended to the Clinical Laboratoryof the Pharmacy College (LACTFAR Federal Universityof Bahia) This population is mainly characterized by low-income individuals who are dependent upon the publichealth system Stool examination was performed by labora-tory staff A flowchart illustrating the study design is providedin Figure 1

23 Sample Selection Two hundred and seventy-threepatients diagnosed with amoeba cysts in their stool werecontacted and invited to participate in the second phase ofthe study A single fresh faecal specimen and 5ml sample ofblood for sera collection were obtained from 90 individualswho agreed to participate in the study Coproantigen andIgG-specific antibodies detection and DNA amplification byPCR were used to diagnose the species of the complex

24 Microscopic Examination Approximately 3 g of faecalmaterial was processed using the formalin-ethyl-acetate cen-trifugation method Morphological analysis was conductedto detect the presence of tetranucleated cysts to confirmthe diagnosis previously established by the spontaneoussedimentation technique A 50120583l portion of the concentratedsample was mixed with iodine spotted on a glass slide andcovered with a coverslip (24 times 24mm) Slides were viewed at40X magnification and results were expressed as a numberof cysts per gram of faeces

25 Parasite Cell Culturing Entamoeba trophozoites wereobtained under xenic conditions in modified Pavlovarsquosmediumwith bacterial flora for coproantigen testing Briefly1 gram of fresh stool was washed five times with distilledwater and 500120583l of sediment was incubated at 37∘Cunder 5CO2in 10ml of Pavlovarsquos medium in round-bottom threaded

culture glass tubes (15 times 15 cm) Amoebas were maintainedwith thrice-weekly subcultures to ensure viability throughthe exponential growth phase The transformation of cystsinto trophozoites as well as trophozoite development andviability was monitored daily for five days

26 E histolytica-Specific Antigen Detection Positive stoolsamples were preserved without fixative and stored at minus20∘Cfor posterior coproantigen testing (galactose adhesin) whichwas performed using the E histolytica II assay (TechLabBlacksburg VA USA) ELISA testing was carried out inaccordance with manufacturerrsquos directions Absorbance at450 nm was measured using a Bio-Rad Model 3550-UVMicroplate Reader (Bio-Rad CA USA)

27 Serological Evaluation The RIDASCREEN Entamoebatest (R-Biopharm AG Darmstadt Germany) was used todetect specific anti-Entamoeba histolytica IgG in human seraThe method utilizes microtiter plates coated with purifiedantigens Briefly serum samples were loaded at 150 in samplediluent and incubated at RT for 15min Following incubationthe microplates were washed with washing buffer to remove

BioMed Research International 3

Laboratory of the Pharmacy College (UFBA)

Microscopic examination

Positive Negative273 (049) 54945 (9951)

in the studyAgreement for participation

Yes90 (33)

No183 (67)

for E histolyticadispar

E histolytica-specific

E histolytica E dispar E moshkovskii

antigen detectionSerological Molecular

characterizationevaluation

moshkovskii complex

55218

Stool samples from individuals attended at Clinical



0 (0) 72 (100) 0 (0)

Amplified Not amplified72 (80) 18 (20)

Figure 1 Flowchart summarizing the study design

any unbound antibodies Protein A conjugate was added toeach well and the microplates were incubated for 15min atRT After five washes the immune complexes were revealedby the addition of urea peroxidase substrate After 15min ofincubation at RT in the dark the reaction was stopped withstop solution and the absorbance at 450 nm was measuredusing a Bio-Rad Model 3550-UV Microplate Reader (Bio-Rad CA USA) Cut-off value have established at OD = 03in accordance with manufacturerrsquos directions

28 Genomic DNA Extraction Positive stool samples werepreserved without fixative and stored at minus20∘C until thetime of DNA extraction Genomic DNA was extracteddirectly from all samples using a QIAmp DNA Mini Kit(QIAGEN CA USA) in accordance with the manufacturerrsquosinstructions Briefly 200mg of stool sample was mixed with14ml ASL buffer in a microcentrifuge tube and vortexeduntil the sample was thoroughly homogenized Followingincubation at 70∘C for 5min samples were centrifuged at25200 g for 1min InhibitEx tablets were subsequently addedto the samples followed by vortexing and centrifugation at

25200 g for 3min Next supernatants were transferred tonew tubes and then proteinase K was added The tubes werereincubated at 70∘C for 10min All samples were then mixedwith ethanol and transferred to spin columns After washingDNA was eluted in 100 120583l elution buffer and immediatelyemployed for PCR analysis

29 Discrimination of E histolytica E dispar and Emoshkovskii Nested multiplex PCR was carried out accord-ing to the protocol described elsewhere [19] using the 16S-like rRNA gene to discriminate between E histolytica Edispar and E moshkovskii The outer primer set E-1 (51015840-TAA GAT GCA CGA GAG CGA AA- 31015840)E-2 (51015840-GTA CAAAGGGCAGGGACGTA-31015840) is specific to a shared fragmentand was specifically designed for all three species The innerprimer pairs EH-1 (51015840-AAG CAT TGT TTC TAG ATC TGAG-31015840)EH-2 (51015840-AAG AGG TCT AAC CGA AAT TAG-31015840)ED-1 (51015840-TCT AAT TCG ATT AGA ACT CT-31015840)ED-2 (51015840-TCC CTA CCT ATT AGA CAT AGC-31015840) and Mos-1 (51015840-GAA CCA AGA GTT TCA CAA AC-31015840)Mos-2 (51015840-CAATAT AAG GCT TGG ATG AT-31015840) are specific to 439-bp


Table 1 Distribution ofE histolyticadisparmoshkovskii complex among individuals attended at the Clinical Laboratory of PharmacyCollege(Salvador Brazil) from February 2010 to June 2014 (n = 55218)

Variables No examined E histolyticadisparmoshkovskii complexNo positive

Age groups (years)le 12 7336 19 7013-18 3205 12 4319-29 9589 54 19830-39 6926 57 20940-49 6964 34 125ge 50 14141 74 271Missing data 7057 23 84Total 55218 273 100GenderMale 15593 117 429Female 32435 144 527lowastMissing data 7190 12 44Total 55218 273 100lowastplt005

bracket for E histolytica 174-bp for E dispar and 553-bp forE moshkovskii respectively Amplification was performed ata total volume of 25120583l containing 03 120583M of each primer255 120583l 10X PCR buffer (200mM Tris-HCl pH 84 500mMKCl) 50mM of each dNTP 25mM of MgCl2 15 U TaqDNA polymerase (Invitrogen Life Technologies CA USA)and 25 120583l of DNA sample An initial DNA amplificationstep was performed using the E-1E-2 primer set in aMyCycler Thermal Cycler (Bio-Rad CA USA) The firstcycle consisting of 2min at 96∘C was followed by 30 cyclesof denaturation for 1min at 92∘C Primers were annealed for1min at 56∘C and extended for 15min at 72∘C An additionalextension step was performed at 72∘C for 7min For nestedamplification 25 120583l of amplicon from the first reaction andprimer sets EH-1EH-2 ED-1ED-2 and Mos-1Mos-2 wereused under identical conditions as those described abovewith the exception of annealing temperature of 48∘C PCRproducts of tested samples and internal controls (DNA of Ehistolytica and E dispar) were analysed by gel electrophoresisDNA fragments were separated on a 10 (wv) agarosegel (Invitrogen Life Technologies CA USA) containing05 120583g ethidium bromideml Gels were photographed underultraviolet illumination (Loccus Biotecnologia SP Brazil)

210 Data Analysis Data were analysed using scatter plotgraphing software (GraphPad Prism v7 CA USA) Descrip-tive statistics are presented as means or medians plusmn standarddeviation or percentages to describe the characteristicsof the studied population including the prevalence of Ehistolytica E dispar and E moshkovskii The Shapiro-Wilktest was employed to assess data normality and Levenersquos testwas used to evaluate the homogeneity of variance Whenthese two assumptions were confirmed ANOVA was usedfor sample comparisons otherwise Fisherrsquos exact test was

employedAll analyseswere two-tailed and a p-value less than5 was considered significant (p lt 005)

3 Results

31 Analysis of Stool Samples The routine parasitologicalanalysis of 55218 stool samples by microscopic examinationidentified E histolytica-like cysts in 273 (sim049)The preva-lence was significantly higher in adults (ge 18 years) Similarlythere was a significant difference in the prevalence of infec-tion betweenmale and female subjects (Table 1) From the 273positive individuals for the E histolyticadisparmoshkovskiicomplex 90 (33) agreed to participate in the survey(Figure 1)

32 E histolytica-Specific Antigen Detection Antigen detec-tion by the E histolytica II assay was employed to identifypositive specimens for E histolytica All 90-stool samplestested were negative for E histolytica which was defined asan optical density value lower than 005 after subtractionof negative control value Additionally it was verified ifthe negative antigen detection was due to the absence oftrophozoites in the well-formed stool Thirty-three sampleswere randomly selected and added to Pavlovarsquos medium forgrowth of Entamoeba species Incubation led to the growthof E histolyticadisparmoshkovskii in 28 (849) culturesFollowing the antigen detection of E histolytica in thetrophozoites transformed in culture all specimens presentednegative results

33 Serological Evaluation Out of 90 samples tested eight(89) were positive and 82 (901) were negative byRIDASCREEN IgG antibody ELISA (Figure 2) Among thepositive no participant was suspected or had hepatic or


06

04

02

00

OD

(450

nm

)

Serum samples(n = 90)

Figure 2 E histolytica-specific IgG detection in 90 positive individuals for E histolyticadisparmoshkovskii complex attended at the ClinicalLaboratory of Pharmacy College (Salvador Brazil) from February 2010 to June 2014 by ELISA The dotted line represents the cut off valueof this test (OD = 03) and samples above this line are considered positive

700 bp500 bp300 bp100 bp

Figure 3 Agarose gel (1) electrophoresis results for amplification of 72 E histolyticadisparmoshkovskii complex positive samples bymultiplex-PCR M molecular weight size marker (100 bp) Lane NC negative control Lane PC positive control for E dispar Lanes 1 3-5 and 7-9 depict the amplification results for E dispar Lanes 2 6 and 10 depict negative results

extrahepatic E histolytica infection and all positive sampleswere positive for E dispar under PCR analysis

34 Molecular Characterization of Entamoeba spp All 90positive faeces samples for E histolyticadisparmoshkovskiicomplex were used in amplified PCR products The 174-bp DNA fragment was successfully amplified in 72 samples(80) indicating positivity for E dispar (Figure 3)MultiplexPCR was capable of identifying the target in 42 samplesdirectly from theDNAHowever 30 samples became positiveonly after successive dilutions (120 to 140) Eighteen samplesremained negative even after diluted to 180 or more Thegeometric mean of the counts of cysts per gram of faeces inamplified samples (390 plusmn 99) was higher than that found innon-amplified samples (196 plusmn 81 p = 002) No PCR productswere detected for E histolytica and E moshkovskii species

4 Discussion

Most of the epidemiological surveys onE histolytica infectionwere conducted before the characterization of the E histolyt-icadisparmoshkovskii complex Accordingly new studies

have been performed to discriminate these species and toestablish the correct distribution of E histolytica infectionworldwide In the present report we employed immunolog-ical and molecular tools to determine the prevalence of Ehistolytica E dispar and E moshkovskii infections amongindividuals attended at the Clinical Laboratory of PharmacyCollege in the city of Salvador (BahiaBrazil)The prevalencerate of E histolyticadisparmoshkovskii complex based onfaecal examination by optical microscopy was around 049This finding suggested a significant reduction in the numberof cases compared to previous studies performed by ourgroup In fact we demonstrated in 2010 prevalence rates of32 and 50 in samples from private and public clinicallaboratories respectively [16 17] This decrease could beexplained in part by the positive impact of government pro-grams which have improved sanitation infrastructure thusreducing the occurrence of intestinal parasites among resi-dents of the Salvador Metropolitan Area [20] Furthermorethe recent expansion of health education and health care ser-vices also may have contributed to the early identification ofE histolytica new cases and prompt treatment Additionallyit appears that E histolytica is more common in North andrare in other regions including Salvador [17 21 22]


The PCR technique presented a sensitivity of 80 andspecificity of 100 Our data are in agreement with our previ-ous report [17] inwhichwe found a sensitivity value of 866In fact 18 DNA samples did not amplify even after successivedilutions It suggests either nondiagnosedE hartmanni infec-tions which could be excluded by morphometric analysisandor PCR [17 23] or nonspecific substances present inthe faecal sediment that inhibited DNA amplification [24]Previous data from our group conducted in Salvador-Bahiashowed that non-amplifiedDNA samples were not associatedwith E hartmanni [17] E histolytica coproantigen detectionrevealed negative results for all assayed samples indicating noE histolytica heterodimer galactoseN-acetyl-galactosamine(GalGalNac) lectin in faecal specimens Significant vari-ability in performance for E histolytica antigen detectionassays in both nonendemic [25 26] and endemic areas[27 28] has been reported Conversely a serological assayproduced eight positive cases (89) suggesting the previousexposure to the parasite All the eight serologically positivesamples rendered negative results for E histolytica by PCRbut positive for E dispar Antibodies remain detectable foryears after successful treatment of E histolytica so it isdifficult to distinguish between active and past infectionreliablyMoreover E histolytica infection confirmed by lectinantigen can occur without antibody detection since theantigens search is more sensitive for E histolytica diagnosis[29]

It is important to note that E dispar has the sametransmission path as other pathogenic protozoa such asE histolytica indicating exposure to faecal contaminationPossibly the production of IgG anti-E histolytica is asso-ciated with immunological memory indicating the parasitecirculation in Salvador Bahia Brazil in accordance with theprevious data of our group [18] Although some reportssuggest a potential role of E dispar in provoking intestinaland extra-intestinal symptoms in humans its pathogenicityremains unclear [30] The prevalence of E dispar is 10 timeshigher than that of E histolyticaworldwide and the attendingphysician must decide if treatment is necessary based moreon clinical evidence [1]

5 Conclusions

In this study the prevalence rate of E histolyticadisparmoshkovskii complex based on faecal examination by opticalmicroscopy was around 049 In the analysed samples bycoproantigen and PCR it was not possible to prove thepresence of E histolytica and E moshkovskii Only E disparwas diagnosed by PCR although the presence of circulatingIgG anti-E histolytica has been detected

Data Availability

The data supporting the conclusions of this article are pro-vided within the article The datasets generated and analysedduring the current study are available from the correspondingauthor upon reasonable request

Conflicts of Interest

The authors declare that they have no conflicts of interest

Acknowledgments

The Goncalo Moniz Institute (Fiocruz-BA) supported thiswork

References

[1] WHOPAHOUNESCO report ldquoA consultation with expertson amoebiasis Mexico City Mexico 28-29 January 1997rdquoEpidemiological Bulletin vol 18 no 1 pp 13-14 1997

[2] L S Diamond and C G Clark ldquoA redescription of entamoebahistolytica schaudinn 1903 (Emended Walker 1911) separatingit from entamoeba dispar brumpt 1925rdquoThe Journal of Eukary-otic Microbiology vol 40 no 3 pp 340ndash344 1993

[3] I K M Ali M B Hossain S Roy et al ldquoEntamoebamoshkovskii infections in children Bangladeshrdquo EmergingInfectious Diseases vol 9 no 5 pp 580ndash584 2003

[4] R D Heredia J A Fonseca and M C Lopez ldquoEntamoebamoshkovskii perspectives of a new agent to be considered in thediagnosis of amebiasisrdquoActa Tropica vol 123 no 3 pp 139ndash1452012

[5] L Rojas P Moran A Valadez et al ldquoEntamoeba histolyticaand Entamoeba dispar infection in Mexican school childrenGenotyping and phylogenetic relationshiprdquo BMC InfectiousDiseases vol 16 no 1 article no 485 2016

[6] M Shibayama S S Dolabella E F Silva and V TsutsumildquoA Brazilian species of Entamoeba dispar (ADO) producesamoebic liver abscess in hamstersrdquo Annals of Hepatology vol6 no 2 pp 117-118 2007

[7] S S Dolabella J Serrano-Luna FNavarro-Garcıa et al ldquoAmoe-bic liver abscess production by Entamoeba disparrdquo Annals ofHepatology vol 11 no 1 pp 107ndash117 2012

[8] C Shimokawa M Kabir M Taniuchi et al ldquoEntamoebamoshkovskii Is Associated With Diarrhea in infants and causesdiarrhea and colitis in micerdquoThe Journal of Infectious Diseasesvol 206 no 5 pp 744ndash751 2012

[9] K Kato T Makiuchi X Cheng and H Tachibana ldquoCom-parison of hemolytic activity of the intermediate subunit ofentamoeba histolytica and entamoeba dispar lectinsrdquo PLoSONE vol 12 no 7 p e0181864 2017

[10] D Talamas-Lara B Chavez-Munguıa A Gonzalez-Robles etal ldquoErythrophagocytosis in Entamoeba histolytica and Enta-moeba dispar a comparative studyrdquo BioMed Research Interna-tional vol 2014 Article ID 626259 10 pages 2014

[11] D Bansal R Sehgal Y Chawla N Malla and R C MahajanldquoMultidrug resistance in amoebiasis patientsrdquo Indian Journal ofMedical Research vol 124 no 2 pp 189ndash194 2006

[12] WHO ldquoAmoebiasisrdquoTheWeekly Epidemiological Record vol 72no 14 pp 97ndash99 1997

[13] F L N Santos ldquoA amebıase deveria detectar-se commetodologias diagnosticas mais economicas e especıficasrdquoSalud(i)Ciencia vol 21 pp 65ndash71 2014

[14] M C Lopez C M Leon J Fonseca et al ldquoMolecularepidemiology of entamoeba first description of entamoebamoshkovskii in a rural area from central colombiardquo PLoS ONEvol 10 no 10 p e0140302 2015


[15] E F F Silva and M A Gomes ldquoEntamoeba histolyticaEntamoeba disparrdquo in Parasitologia Humana D P Neves A Lde Melo M Linardi and A Vitor Eds p 264 Atheneu PressSao Paulo Brazil 2016

[16] L P Santos F L Neves Santos and N M Soares ldquoPrevalenciade parasitoses intestinais em pacientes atendidos no HospitalUniversitario Professor Edgar Santos Salvador-Bahiardquo Revistade Patologia Tropical vol 36 no 3 pp 237ndash246 2007

[17] F L Santos M d Goncalves and N M Soares ldquoValidationand utilization of PCR for differential diagnosis and prevalencedetermination of Entamoeba histolyticaEntamoeba dispar inSalvador City Brazilrdquo The Brazilian Journal of Infectious Dis-eases vol 15 no 2 pp 119ndash125 2011

[18] R K Silva F T Pacheco A S Martins et al ldquoPerformanceof microscopy and ELISA for diagnosing Giardia duodenalisinfection in different pediatric groupsrdquo Parasitology Interna-tional vol 65 no 6 pp 635ndash640 2016

[19] K Khairnar and S C Parija ldquoA novel nested multiplex poly-merase chain reaction (PCR) assay for differential detection ofEntamoeba histolytica E moshkovskii and E dispar DNA instool samplesrdquo BMCMicrobiology vol 7 no 1 p 47 2007

[20] M L Barreto B Genser A Strina et al ldquoEffect of city-widesanitation programme on reduction in rate of childhood diar-rhoea in northeast Brazil assessment by two cohort studiesrdquoThe Lancet vol 370 no 9599 pp 1622ndash1628 2007

[21] M Benetton A Goncalves M Meneghini E Silva and MCarneiro ldquoRisk factors for infection by the Entamoeba histolyt-icaE dispar complex An epidemiological study conductedin outpatient clinics in the city of Manaus Amazon RegionBrazilrdquo Transactions of the Royal Society of Tropical Medicineand Hygiene vol 99 no 7 pp 532ndash540 2005

[22] L L Braga M L Gomes M W Silva C Paiva A Salesand B J Mann ldquoEntamoeba histolytica and Entamoeba disparinfections as detected by monoclonal antibody in an urbanslum in Fortaleza Northeastern Brazilrdquo Journal of the BrazilianSociety of Tropical Medicine vol 34 no 5 pp 467ndash471 2001

[23] D A Calegar B C Nunes K J L Monteiro et al ldquoFrequencyandmolecular characterisation of Entamoeba histolytica Enta-moeba dispar Entamoeba moshkovskii and Entamoeba hart-manni in the context of water scarcity in northeastern BrazilrdquoMemorias do Instituto Oswaldo Cruz vol 111 no 2 pp 114ndash1192016

[24] J J Verweij J Blotkamp E A Brienen A Aguirre and AM Polderman ldquoDifferentiation of entamoeba histolytica andentamoeba dispar cysts using polymerase chain reaction onDNA isolated from faeces with spin columnsrdquo European Journalof Clinical Microbiology amp Infectious Diseases vol 19 no 5 pp358ndash361 2000

[25] L G Visser J J Verweij M Van Esbroeck W M EdelingJ Clerinx and A M Polderman ldquoDiagnostic methods fordifferentiation of Entamoeba histolytica and Entamoeba disparin carriers Performance and clinical implications in a non-endemic settingrdquo International Journal of Medical Microbiologyvol 296 no 6 pp 397ndash403 2006

[26] D Stark S van Hal R Fotedar et al ldquoComparison of stoolantigen detection kits to PCR for diagnosis of amebiasisrdquoJournal of Clinical Microbiology vol 46 no 5 pp 1678ndash16812008

[27] S RoyM Kabir DMondal I K AliW A Petri and R HaqueldquoReal-Time-PCR assay for diagnosis of entamoeba histolyticainfectionrdquo Journal of Clinical Microbiology vol 43 no 5 pp2168ndash2172 2005

[28] M R Gaafar ldquoEvaluation of enzyme immunoassay techniquesfor diagnosis of the most common intestinal protozoa in fecalsamplesrdquo International Journal of Infectious Diseases vol 15 no8 pp e541ndashe544 2011

[29] R Haque N U Mollah I K M Ali et al ldquoDiagnosis ofamebic liver abscess and intestinal infection with the TechLabEntamoeba histolytica II antigen detection and antibody testsrdquoJournal of Clinical Microbiology vol 38 no 9 pp 3235ndash32392000

[30] F M S Oliveira E Neumann M A Gomes et al ldquoEntamoebadispar Could it be pathogenicrdquo Tropical Parasitology vol 5 no1 pp 9ndash14 2015

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for preventing unnecessary and indiscriminate treatmentwith anti-amoebic chemotherapy which could lead to drugresistance [11] The World Health Organization advises thatcases determined to have E histolytica should be treatedwhether or not clinical symptoms are present [12] Addi-tionally control measures could be more efficiently appliedin geographical areas with well-known epidemiological set-tings

Regarding the low viability of trophozoites in diarrhealspecimens and irregular faecal cyst excretion in asymp-tomatic hosts the use of different diagnostic methods isrequired to increase the sensitivity of parasite identificationin faecal samples Immunoassays for coproantigen detectionof specific E histolytica-GalGalNAc lectin have been usedas alternative methods for the diagnosis of amebiasis [13]Molecular techniques based on the amplification of parasiteDNA have been shown to be a highly sensitive and specificmethod that allows the detection of different Entamoebaspecies [14] However a negative result does not rule outthe presence of the parasite due to interference from PCRinhibitors present in faeces that may hamper DNA amplifi-cation

In Brazil due to regional differences in sanitation andsocioeconomic conditions E histolyticadisparmoshkovskiicomplex distribution is irregular with 25-11 in the Southand Southeast 19 in the North and the Amazon region andapproximately 10 in the Northeast and Central West [15]In the city of Salvador the capital of the Brazilian state ofBahia the presence of the complexwas shown in some studiesconducted by our group with a prevalence of 5 and 32 inindividuals attended at public [16] and the private health sys-tem respectively Moreover 15 (2621788) of positive stoolsamples for E histolyticaE dispar complex was analysed byPCR and no DNA amplified for E histolytica DNA amplifiedfor E dispar was observed in 866 of samples (227262) [17]However in a previous study also conducted by our group46 (7153) of hospitalized diarrheal children from Salvadortested positive for specific lectin of E histolytica-GalGalNAc[18]

These findings further support evidence that E histolyticamay be infecting humans in Salvador Given the importanceof the actual prevalence of E histolytica E dispar andE moshkovskii infections this study aimed to assess thefrequency of each species by molecular and immunologicaltechniques in individuals attended at the public health systemin Salvador-Bahia Brazil

2 Materials and Methods

21 Ethical Considerations Approval was granted by theInstitutional Review Board (IRB) for Human Research atthe Goncalo Moniz Institute Oswaldo Cruz Foundation(FIOCRUZ) Salvador Bahia-Brazil registration number1002006 All procedures were conducted in strict adherenceto the principles laid out in the Declaration of HelsinkiInformed consentwas obtained fromparticipantswho agreedto participate of the study Samples were anonymously codedto protect each participantrsquos identity

22 Study Design and Population A cross-sectional studywas performed from February 2010 to June 2014 involving55218 individuals who attended to the Clinical Laboratoryof the Pharmacy College (LACTFAR Federal Universityof Bahia) This population is mainly characterized by low-income individuals who are dependent upon the publichealth system Stool examination was performed by labora-tory staff A flowchart illustrating the study design is providedin Figure 1

23 Sample Selection Two hundred and seventy-threepatients diagnosed with amoeba cysts in their stool werecontacted and invited to participate in the second phase ofthe study A single fresh faecal specimen and 5ml sample ofblood for sera collection were obtained from 90 individualswho agreed to participate in the study Coproantigen andIgG-specific antibodies detection and DNA amplification byPCR were used to diagnose the species of the complex

24 Microscopic Examination Approximately 3 g of faecalmaterial was processed using the formalin-ethyl-acetate cen-trifugation method Morphological analysis was conductedto detect the presence of tetranucleated cysts to confirmthe diagnosis previously established by the spontaneoussedimentation technique A 50120583l portion of the concentratedsample was mixed with iodine spotted on a glass slide andcovered with a coverslip (24 times 24mm) Slides were viewed at40X magnification and results were expressed as a numberof cysts per gram of faeces

25 Parasite Cell Culturing Entamoeba trophozoites wereobtained under xenic conditions in modified Pavlovarsquosmediumwith bacterial flora for coproantigen testing Briefly1 gram of fresh stool was washed five times with distilledwater and 500120583l of sediment was incubated at 37∘Cunder 5CO2in 10ml of Pavlovarsquos medium in round-bottom threaded

culture glass tubes (15 times 15 cm) Amoebas were maintainedwith thrice-weekly subcultures to ensure viability throughthe exponential growth phase The transformation of cystsinto trophozoites as well as trophozoite development andviability was monitored daily for five days

26 E histolytica-Specific Antigen Detection Positive stoolsamples were preserved without fixative and stored at minus20∘Cfor posterior coproantigen testing (galactose adhesin) whichwas performed using the E histolytica II assay (TechLabBlacksburg VA USA) ELISA testing was carried out inaccordance with manufacturerrsquos directions Absorbance at450 nm was measured using a Bio-Rad Model 3550-UVMicroplate Reader (Bio-Rad CA USA)

27 Serological Evaluation The RIDASCREEN Entamoebatest (R-Biopharm AG Darmstadt Germany) was used todetect specific anti-Entamoeba histolytica IgG in human seraThe method utilizes microtiter plates coated with purifiedantigens Briefly serum samples were loaded at 150 in samplediluent and incubated at RT for 15min Following incubationthe microplates were washed with washing buffer to remove






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Yes90 (33)

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moshkovskii complex

55218




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3 Results





06

04

02

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OD

(450

nm

)



700 bp500 bp300 bp100 bp




4 Discussion






5 Conclusions


Data Availability




Acknowledgments


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Volume 2018

Zoology











Volume 2018

















Advances in




Enzyme Research





Volume 2018









3 Results





06

04

02

00

OD

(450

nm

)



700 bp500 bp300 bp100 bp




4 Discussion






5 Conclusions


Data Availability




Acknowledgments


References


































Volume 2018

Zoology











Volume 2018

















Advances in




Enzyme Research





Volume 2018



06

04

02

00

OD

(450

nm

)



700 bp500 bp300 bp100 bp




4 Discussion






5 Conclusions


Data Availability




Acknowledgments


References


































Volume 2018

Zoology











Volume 2018

















Advances in




Enzyme Research





Volume 2018





5 Conclusions


Data Availability




Acknowledgments


References


































Volume 2018

Zoology











Volume 2018

















Advances in




Enzyme Research





Volume 2018





















Volume 2018

Zoology











Volume 2018

















Advances in




Enzyme Research





Volume 2018


a cross-sectional study of entamoeba histolytica/dispar...

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