genetic diversity and distribution of blastocystis subtype...

Research ArticleGenetic Diversity and Distribution of BlastocystisSubtype 3 in Human Populations with Special Referenceto a Rural Population in Central Mexico

Liliana Rojas-Velaacutezquez 12 Patricia Moraacuten 1 Angeacutelica Serrano-Vaacutezquez1

Leonardo D Fernaacutendez 3 Horacio Peacuterez-Juaacuterez12 Augusto C Poot-Hernaacutendez4

Tobiacuteas Portillo 5 Enrique Gonzaacutelez1 Eric Hernaacutendez1 Oswaldo Partida-Rodriacuteguez 1

Miriam E Nieves-Ramiacuterez1 Ulises Magantildea12 Javier Torres 6 Luis E Eguiarte7

Daniel Pintildeero7 and Cecilia Ximeacutenez 1

1Unidad de Investigacion en Medicina Experimental Facultad de Medicina Universidad Nacional Autonoma de Mexico (UNAM)Dr Balmis 148 Doctores Cuauhtemoc 06726 Ciudad de Mexico Mexico2Unidad de Posgrado Universidad Nacional Autonoma de Mexico (UNAM) Circuito de Posgrado SN Coyoacan Cd Universitaria04510 Ciudad de Mexico Mexico3Centro de Investigacion en Recursos Naturales y Sustentabilidad (CIRENYS) Universidad Bernardo OrsquoHigginsAvenida Viel 1497 Santiago Chile4Departamento de Ingenierıa de Sistemas Computacionales y AutomatizacionSeccion de Ingenierıa de Sistemas Computacionales Instituto de Investigaciones en Matematicas Aplicadas y en SistemasUniversidad Nacional Autonoma de Mexico (UNAM) Circuito Escolar 3000 Cd Universitaria Coyoacan04510 Ciudad de Mexico Mexico5Unidad de Bioinformatica Bioestadıstica y Biologıa Computacional Red de Apoyo a la InvestigacionCoordinacion de la Investigacion Cientıfica UNAM Instituto Nacional de Ciencias Medicas y NutricionVasco de Quiroga 15 Tlalpan 14080 Ciudad de Mexico Mexico6Unidad de Investigacion Medica en Enfermedades Infecciosas y Parasitarias Hospital de Pediatrıa Centro Medico Siglo XXIInstituto Mexicano del Seguro Social (IMSS) Avenida Cuauhtemoc 330 Doctores Cuauhtemoc 06720 Ciudad de Mexico Mexico7Departamento de Ecologıa Evolutiva Instituto de Ecologıa Universidad Nacional Autonoma de Mexico (UNAM)Circuito Exterior SN Junto al Jardın Botanico Cd Universitaria Coyoacan 04510 Ciudad de Mexico Mexico

Correspondence should be addressed to Liliana Rojas-Velazquez lhilyyahoocom and Cecilia Ximenez cximenezunammx

Received 29 December 2017 Accepted 11 February 2018 Published 18 March 2018

Academic Editor Daniele Corsaro

Copyright copy 2018 Liliana Rojas-Velazquez et alThis is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited

Blastocystis subtype 3 (ST3) is a parasitic protist found in the digestive tract of symptomatic and asymptomatic humans around theworld While this parasite exhibits a high prevalence in the human population its true geographic distribution and global geneticdiversity are still unknown This gap in knowledge limits the understanding of the spread mechanisms epidemiology and impactthat this parasite has on human populations Herein we provided new data on the geographical distribution and genetic diversityof Blastocystis ST3 from a rural human population in Mexico To do so we collected and targeted the SSU-rDNA region in fecalsamples from this population and further compared its genetic diversity and structure with that previously observed in populationsof Blastocystis ST3 from other regions of the planet Our analyses reveled that diversity of Blastocystis ST3 showed a high haplotypediversity and genetic structure to theworld level however theywere low in theMorelos populationThehaplotype network revealeda common widespread haplotype from which the others were generated recently Finally our results suggested a recent expansionof the diversity of Blastocystis ST3 worldwide

HindawiBioMed Research InternationalVolume 2018 Article ID 3916263 7 pageshttpsdoiorg10115520183916263

2 BioMed Research International

1 Introduction

Blastocystis (Heterokonta Stramenopiles) is a genus com-prising parasitic protists that inhabit the digestive tractof several metazoans such as fishes amphibians birdsreptiles rodents and humans [1ndash3] Blastocystis is globallydistributed showing a high rate of infection from underde-veloped to developed countries [4 5]

This parasite is often transmitted via the oral-fecal routeto people who work directly with animals such as thoseinvolved in intensive animal farming or industrial livestockproduction [6] In humans the signs and symptoms asso-ciated with Blastocystis infection range from diarrhea toflatulence bloating and abdominal discomfort [7 8] withthe ldquoirritable bowel syndromerdquo (IBS) being the most frequentclinical manifestation [8ndash11]

Molecular evidence based on the small subunit ribosomalRNA (SSU-rDNA) gene suggests that at least 17 geneticsubtypes can be recognized within Blastocystis [12] Nineof these subtypes are found in humans with subtype 3(ST3 hereafter) being the most common in epidemiologicalstudies worldwide [12ndash18] ST3 has been regarded to triggerIBS in humans [8] and recent research also suggests anassociation between this subtype and colorectal cancer [19]Other studies however suggest a lack of association betweenthe ST3 and some type of symptomatology in humans [7 20]While Blastocystis ST3 has medical importance and highprevalence in humans the realmagnitude of its genetic diver-sity and geographical distribution remains so far unknown[5 17] Apparently the ST3 exhibits a broader geographicaldistribution and higher genetic diversity than other geneticsubtypes of Blastocystis [16 21] but this hypothesis still needsto be tested using genetic data and clinical cases from bothwell-studied and undersampled geographical areas In thiscontext there are very few geographical and genetic data onBlastocystis ST3 fromMexico

Herein we aimed to provide newdata on the geographicaldistribution and genetic diversity of Blastocystis ST3 from arural and asymptomatic human population in Mexico Todo so we collected and targeted the SSU-rDNA region infecal samples from this population and further comparedits genetic diversity and structure with those previouslyobserved in populations of Blastocystis ST3 from otherregions of the planet

2 Materials and Methods

21 Ethical Considerations The protocol used in this studywas conducted under the ethical principles and approval ofboth the Mexican Commission on Ethics and Research ofthe Health Ministry of the State of Morelos (Comisiones deEtica y de Investigacion del Ministerio de Salud del Estado deMorelos) and the Commission on Ethics in Research of theFacultad deMedicina of theUniversidadNacional Autonomade Mexico (UNAM) (Comite de Etica de Investigacion de laFacultad de Medicina de la Universidad Nacional Autonomade Mexico) The guidelines of the committees are basedon the Mexican Official Norm (Norma Oficial Mexicana

NOM-012-SSA3-2007) which regulates the ethical principlesof every research on humans and on laboratory animalsas well as on the Declaration of Helsinki which set ethicalprinciples regarding human experimentation developed bythe World Health Organization (WHO)

Based on the abovementioned guidelines our studyonly used samples from volunteers who were respectivelyinformed about the objectives of this research the potentialrisks (if any) and the sampling procedures We obtained aninformed consent letter from all the participants

22 Sampling and Analysis Between May and November2015 fecal samples were collected from 182 volunteers (86males and 96 females) fromPuente de Ixtla in the communityof Xoxocotla State of Morelos (Mexico) ranging in agefrom 2 to 51 years old The asymptomatic status was definedaccording to the ROME III criteria Three fecal samples werecollected from each volunteer on three consecutive daysThe samples were maintained at 4∘C and transported to thelaboratory in Mexico City on the same day of collection Asubsample of each fecal sample was smeared stained with4 Lugolrsquos iodine solution and examined under a lightmicroscope at 10x and 40x magnifications [22]

23 Amplification and Sequencing of SSU-rDNA DNA wasextracted from fresh fecal samples using QIAamp DNAstool kit (QIAGEN Hilden Germany) and following themanufacturerrsquos instructions PCR protocol targeting the SSU-rDNA was conducted according to Scicluna et al [23] Inbrief we used a total mixture of 20120583l 20120583Mof primers RD5(51015840-ATC TGG TTG ATC CTG CCAG T-31015840) and BhRDr (51015840-GAG CTT TTT AAC TGC AAC AAC G-31015840) [23] as wellas 0025U of polymerase (AmpliTaq Platinum PolymeraseInvitrogen) To verify the presence of a single band andthe size of the amplified products (approximately 600 bp)the PCR products were separated by electrophoresis inagarose gel (15) in the presence of ethidium bromide visu-alized by ultraviolet transillumination and photographedThe amplification product of a 600 bp fragment of theBlastocystis SSU-rDNA was purified and sequenced using adideoxynucleotide-terminalmethod Sequencingwas carriedout in a capillary sequencer (ABI-Avant 100 University ofWashington) The sequences obtained were edited andoranalyzed with BioEdit MEGA 50 software [24 25] and adhoc scripts from Python These sequences were comparedto sequences available in GenBank employing BLAST toestablish their identityThe final sequences were deposited inGenBank under accession numbers MF539962ndashMF540015

24 Global Genetic Diversity and Haplotype Network forBlastocystis ST3 We investigated the global genetic diversity(ie LatinAmerica Europe andAsia) withinBlastocystis ST3using the novel SSU-rDNA sequences reported in the presentstudy and those previously reported within the literature Weprovided an exhaustive list of the latter sequences (119899 = 169)and sources in the Supplementary Material (available here)We investigated the following descriptive statistics of genetic

BioMed Research International 3

Mic

rosc

opic

anal

ysis

Bsp

Bsp + OP

OP

OP = other parasites

Negative

67

207

5

Figure 1 Frequency of intestinal parasites based on microscopicanalysis of the fecal samples taken in Morelos Mexico Blastocystiswas the only parasitic infection found in 67 of individualsand in 7 in coinfection with other parasites Bsp BlastocystisOP parasites other than Blastocystis BSP + OP coinfection ofBlastocystis and other parasites Negative no parasite found AmongOP Chm Chilomastix mesnili Ec Entamoeba coli En Endolimaxnana Hn Hymenolepis nana Gl Giardia lamblia Ib Iodamoebabutschlii

diversity using the software DnaSP ver 51001 [26] numberof segregating sites (119878) number of haplotypes (ℎ) haplotypediversity (Hd) and nucleotide diversity (120587) for each set ofsequences according to their geographical region of originWe built a global haplotype network using TCS networkinference method [27] implemented in the PopART pro-gram ver 17 (httppopartotagoacnzdownloadsshtml) toinvestigate the global genealogical relationship between thedifferent haplotypes of Blastocystis ST3 We also ran Tajimarsquos119863 test in the software DnaSP [26] to investigate possibleevents of global population expansion on Blastocystis ST3We finally estimated pairwise 119865ST statistics in the softwareArlequin ver 311 (httpcmpgunibechsoftwarearlequin3)to investigate whether the geographical populations of Blas-tocystis ST3 were genetically structured

3 Results

31 Frequency of Blastocystis ST3 in Morelos Mexico A mi-croscopic analysis revealed that 148 (8132) of the 182 fecalsamples collected inMorelos (Mexico) exhibited at least sometype of intestinal parasite These 148 samples (positive sam-ples hereafter) harbored different parasites including repre-sentatives ofBlastocystis Chilomastixmesnili Entamoeba coliHymenolepis nana Iodamoeba butschlii Endolimax nanathe Entamoeba histolyticaEntamoeba dispar complex andGiardia lamblia Among the abovementioned parasites Blas-tocystis had the greatest frequency occurring in 109 (74)out of 148 positive samples It was also the unique parasite in99 (67) out of 148 positive samples and 7 of the samples(10148) were coinfected with other parasites (Figure 1)

Subt

ypes

15

75

9

ST1

ST2

ST3

Figure 2 Frequency of Blastocystis subtypes in the study popula-tion Targeting the SSU-rDNA according to DNA-barcoding ThreeBlastocystis subtypes (ST) were recorded according to the followingfrequencies Blastocystis ST1 97 (119899 = 7 samples) ST2 153(119899 = 11 samples) and ST3 75 (119899 = 54 samples)

Further PCR and sequencing procedures successfullyconfirmed the presence of three different Blastocystis sub-types in 72 of the 148 positive samples collected in MorelosThese three Blastocystis subtypes (ST) were recorded accord-ing to the following frequencies Blastocystis ST1 97 (119899 = 7samples) ST2 153 (119899 = 11 samples) and ST3 75 (119899 = 54samples) (Figure 2)

32 Genetic Diversity of ST3 andHaplotype Network Geneticdiversity indices revealed a total of 44 segregating sites (119878) and20 haplotypes (ℎ) as well as a total haplotype diversity (Hd)of 0563 and nucleotide diversity (120587) of 0019 Tajimarsquos 119863 testprovided values ranging between minus1303 and minus2363 (Table 1)A pairwise 119865st analysis revealed that there is very lowgenetic differentiation between all geographical populationsof Blastocystis ST3 (Table 2)

The number of haplotypes ranged from 3 to 15 betweenhuman populations the number of segregating sites rangedbetween 1 and 35 haplotype diversity ranged between 0142and 0740 and nucleotide diversity ranged between 0001 and0045 (Table 1) The ST3 genetic diversity of Latin Americanpopulations (exceptMorelosrsquos population) and Eurasia exhib-ited the highest values of genetic diversity indices in contrasttoMorelosrsquos population where low haplotype diversity (threehaplotypes) was detected (Table 1)

The haplotype network showed the haplotype distri-bution of the ST3 (Figure 3) In general the worldwidehaplotype network evidenced large levels of diversity with atotal of 20 haplotypes and haplotype 1 was the dominantThenetwork showed a star topology radial distribution (Figure 3)Also haplotype 1 was the most frequently found in Morelosrsquospopulation and this haplotype is commonly distributed inAmerican populations

4 Discussion

In the present study we analyzed the frequency and distribu-tion of Blastocystis subtypes in an asymptomatic rural pop-ulation The results revealed a great frequency of Blastocystis


Table 1 Statistics data of genetic diversity observed within different geographical populations of Blastocystis ST3 around the world

Populations 119873 119878 ℎ Hd 120587 plusmnSD Tajimarsquos119863Morelos 54 1 3 014186 000107 0068 minus168258ns

Latin America 69 25 15 067775 001334 0058 minus236261lowastlowast

Eurasia 46 35 11 074010 004473 0052 minus130307ns

All populations 169 44 20 056276 001886 0044 minus220000lowastlowast

119873 number of sequences 119878 number of segregating sites ℎ number of haplotypes Hd haplotype diversity 120587 nucleotide diversity ns not significant lowastlowast119901 lt001 Latin America Blastocystis populations of North and South America (ie Mexico Colombia Brazil Ecuador Bolivia Peru and Argentina) except thatof Morelos Eurasia Blastocystis populations of Europa and Asia (ie Nepal Switzerland Iraq Italy and France)

Table 2 Estimates of 119865ST based on the SSU-rDNA variationobserved between different geographical populations of the parasiteBlastocystis ST3

Population Morelos Latin America EurasiaMorelos ------Latin America 004165ns ------Eurasia 009164ns 005975ns ------Latin America Blastocystis populations of North and South America (ieMexico Colombia Brazil Ecuador Bolivia Peru and Argentina) exceptthat of Morelos Eurasia Blastocystis populations of Europe and Asia (ieNepal Switzerland Iraq Italy and France) Probability obtained by apermutation test with 50000 replicates ns not significant

from 74 above the national average as the frequency of thisparasite varies from 23 to 61 inMexico [20 28 29] Recentstudies in South America have described similar frequencyof Blastocystis in the human population (21 to 67) [30ndash32] Around the world Blastocystis exhibits a frequency rangeof 05 to 62 [4] The higher prevalence of Blastocystis hasbeen linked to hygiene factors including the consumptionof foodwater contaminated with Blastocystis and exposureto domestic and peridomestic animals infected too with thisparasite [4 33]

Many years ago Blastocystis was considered as sapro-phytic yeast of the digestive tract innocuous for the host[34] Nowadays we can observe that this parasite is widelydistributed in the human population in the world and itis similarly distributed in symptomatic and asymptomaticindividuals For instance Morelosrsquos population (Mexico)showed a high infection frequency of Blastocystis althoughthe participants were asymptomatic suggesting tolerance tothis parasite as reported elsewhere [20 35 36]

Blastocystis has a high worldwide genetic diversity repre-sented by 17 subtypes (ST1ndashST17) [5] It is possible that thereare other subtypes capable of infecting humans and othervertebrates [4 5] Regarding the distribution of subtypesin the present study ST1 (97) ST2 (153) and ST3(75) were identified among 72 human isolates successfullygenotyped Globally Blastocystis ST3 is the most prevalentsubtype in humans found in different geographic areas [5 37]In our study the frequency of ST3was 75 high compared toother populations ofMexico as the state ofMichoacan wherethe frequency of this subtype was 21 and in Mexico City itwas 42 [38 39]

In Latin America the frequency of ST3 is high [4 37]with the following frequencies reported forBlastocystis ST3 in

H1

H3H12

H7

H11

H8

H9

H18

H14

H13

H15H4

H20

H19

H17

H10

H2

H5

H6 H16

Morelos

Latin AmericaEurasia

13 samples

1 sample

Figure 3 Haplotype network of Blastocystis ST3 of human popu-lations at different regions from Latin America Europe and AsiaEach circle represents a haplotype and each color represents theplace where it was obtained The size of each circle is proportionalto the frequency of the haplotype in each population where it wasfoundThe circles in black stand formissing haplotypes and the shortlines show the mutational steps

this political region 14 in Colombia 36 in Brazil 84 inEcuador 30 in Bolivia 92 in Peru and 63 in Argentina[40] While ST3 is amply distributed worldwide it is moreprevalent in Latin America [37] which opens the possibilitythat this subtype was generated in this geographic area andspread to the rest of the continents

To improve our knowledge on the magnitude of thegenetic diversity ofBlastocystis ST3 on the planet we analyzedsequences of Mexico City South America France Switzer-land Italy Nepal and Iraq and the sequences obtained in thepresent study (169 total sequences 54 from Morelos and 115from the NCBI database) The genetic parameters calculatedfor these sequences suggested a recent population increase ordirectionalpurifying selection These results are supportedby the haplotype network which showed a star topologywith the haplotypes distributed in a radial way supportinginferences of a recent geographical expansion in Blastocystis


ST3 This behavior is similar to reporting by other parasitessuch as Plasmodium falciparum [41]

A total of 20 haplotypes were found on all sequencesanalyzed Haplotype 1 was the most abundant and widelydistributed being detected in the majority of the studiedcountriesmainly in LatinAmericaThese results are showingthat haplotype 1 is perhaps an ancestral type from which allthe other haplotypes have been generated recently Haplotype1 probably originated in Latin America and has recentlycolonized other regions of the world probably via humanmigration [42]

In some studies it has been observed that this subtype hasbeen strongly related to rural populations [8 43] It is possibleto hypothesize that the migration phenomenon [42] occursmainly from the displacement of these rural communitiesinfectedwithBlastocystis ST3 haplotype 1 spreading this para-site into the cities Already in large cities the transmission ofBlastocystis ST3 haplotype 1 could happen from one personto another [43] In addition due to the opening of politicalborders between developed countries this process of travel-associated infection is common because travelers may act ascarriers of this parasite [44]

Also it is possible that the other haplotypes of BlastocystisST3 could be colonizing different areas promoted by thehuman migratory phenomenon This idea would explainthe high representation of certain haplotypes throughoutthe American continent and others in European countriesFor example seven sequences of haplotype 9 correspondto South America and only one corresponds to Europe(Switzerland)

However to test both hypotheses it will be necessary tohave a larger number of sequences and complete genomesfrom around the world It is known that migration causesmobility of parasites and it is important to have informationof other sociodemographic parameters of the host as thenationality or antecedents of previous travels to endemicareas [45] In addition the distribution of Blastocystis couldbe related to the lack of symptoms that occurs in many casesbecause asymptomatic Blastocystis infections are not treatedand therefore there are a large number of carriers of thisparasite It is known that these factors can influence thedistribution of parasites [42 46]

With respect to Morelos all the sequences of BlastocystisST3 were grouped into three haplotypes which means thatthere is a low genetic diversity a reduced rate of mutationsand little genetic differentiation this suggests isolation andhomogeneity in the population

5 Conclusions

To our knowledge this is the first study analyzing the haplo-type diversity and distribution of Blastocystis ST3 subtypes indifferent human populations In addition our work facilitatesthe vision of a global distribution in Blastocystis ST3 Weprovide evidence of a recent expansion of this subtype thatmay be related to the migration of humans to other regionsof the world However it is necessary to continue studyingthis parasite in order to generate amore complete knowledgethat allows us to know the course of Blastocystis infection

its epidemiology and the causal factors that contribute to itsdispersion dynamics and distribution

Disclosure

Liliana Rojas-Velazquez is a doctoral student from Programade Doctorado en Ciencias Biomedicas Universidad NacionalAutonoma de Mexico (UNAM) and received Fellowship348424239901 from CONACYT This paper constitutes apartial fulfillment of the Graduate Program

Conflicts of Interest

The authors declare that there are no conflicts of interestregarding the publication of this paper

Acknowledgments

The present work was supported by Grants IN218214 andIN-226511 from PAPIIT (DGAPA) Universidad NacionalAutonoma de Mexico (UNAM) CONACyT 210-C01-140990from the National Council for Science and Technologyin Mexico (CONACyT) and FISIMSSProt699 from theMexican Institute of Social Security (IMSS) The authorswould like to appreciate the collaboration of the HealthMinistry of the State of Morelos Mexico for authoriz-ing the establishments of the center of activity in thefacilities and in the community of Xoxocotla They alsoacknowledge the technical support provided by MS MarthaZaragoza the Chemist Angeles Padilla and Alejandro FloresThey also highly appreciate the informatics assistance ofAngelica Serrano-Ahumada and Marco Gudino LeonardoD Fernandez is supported by CONICYT (FONDECYTProject no 11170927) and byUniversidadBernardoOrsquoHiggins(Project UBOVRIP170201)

Supplementary Materials

List of the sequences reported in the present study and thosepreviously reported within the literature (SupplementaryMaterials)

References

[1] H Yoshikawa Z Wu J Howe T Hashimoto N Geok-Chooand K S W Tan ldquoUltrastructural and phylogenetic studies onBlastocystis isolates from cockroachesrdquo Journal of EukaryoticMicrobiology vol 54 no 1 pp 33ndash37 2007

[2] J D Silberman M L Sogin and D D Leipe ldquoHuman parasitefinds taxonomic homerdquo Nature vol 380 no 6573 p 398 1996

[3] A Stechmann K Hamblin V Perez-Brocal et al ldquoOrganellesin blastocystis that blur the distinction between mitochondriaand hydrogenosomesrdquo Current Biology vol 18 no 8 pp 580ndash585 2008

[4] C G Clark M van der Giezen M A Alfellani and CR Stensvold Recent Developments in Blastocystis ResearchElsevier Amsterdam Netherlands 2013


[5] M A Alfellani D Taner-Mulla A S Jacob et al ldquoGeneticdiversity of blastocystis in livestock and zoo animalsrdquo Protistvol 164 no 4 pp 497ndash509 2013

[6] K S W Tan ldquoNew insights on classification identificationand clinical relevance of Blastocystis spprdquo Clinical MicrobiologyReviews vol 21 no 4 pp 639ndash665 2008

[7] C V Barbosa R D J Batista R P Igreja C M D Levy H WD Macedo and H L C Santos ldquoDistribution of Blastocystissubtypes isolated from humans from an urban community inRio de Janeiro Brazilrdquo Parasites and Vectors vol 10 no 1 p 5182017

[8] A A El-Badry W M Abd ElWahab D A Hamdy and AAboud ldquoBlastocystis subtypes isolated from irritable bowelsyndrome patients and co-infection with Helicobacter pylorirdquoParasitology Research pp 1ndash11 2017

[9] A Giacometti O Cirioni A Fiorentini M Fortuna and GScalise ldquoIrritable bowel syndrome in patients with Blastocystishominis infectionrdquo European Journal of Clinical Microbiology ampInfectious Diseases vol 18 no 6 pp 436ndash439 1999

[10] J YakoobW JafriM A Beg et al ldquoIrritable bowel syndrome isit associated with genotypes of blastocystis hominisrdquo Parasitol-ogy Research vol 106 no 5 pp 1033ndash1038 2010

[11] K A Jadallah L F Nimri and R A Ghanem ldquoProtozoan par-asites in irritable bowel syndrome a case-control studyrdquoWorldJournal of Gastrointestinal Pharmacology and Therapeutics vol8 no 4 pp 201ndash207 2017

[12] C R Stensvold G K Suresh K S W Tan et al ldquoTerminologyfor Blastocystis subtypes - a consensusrdquo Trends in Parasitologyvol 23 no 3 pp 93ndash96 2007

[13] F Dogruman-Al S Kustimur H Yoshikawa et al ldquoBlastocystissubtypes in irritable bowel syndrome and inflammatory boweldisease in Ankara Turkeyrdquo Memorias do Instituto OswaldoCruz vol 104 no 5 pp 724ndash727 2009

[14] A Moosavi A Haghighi E N Mojarad et al ldquoGeneticvariability of Blastocystis sp Isolated from symptomatic andasymptomatic individuals in Iranrdquo Parasitology Research vol111 no 6 pp 2311ndash2315 2012

[15] H Yoshikawa Z Wu I Kimata et al ldquoPolymerase chainreaction-based genotype classification among human Blasto-cystis hominis populations isolated from different countriesrdquoParasitology Research vol 92 no 1 pp 22ndash29 2004

[16] C R Stensvold M Alfellani and C G Clark ldquoLevels of geneticdiversity vary dramatically between Blastocystis subtypesrdquoInfection Genetics and Evolution vol 12 no 2 pp 263ndash2732012

[17] M A Alfellani C R Stensvold A Vidal-Lapiedra E S UOnuoha A F Fagbenro-Beyioku and C G Clark ldquoVariablegeographic distribution of Blastocystis subtypes and its poten-tial implicationsrdquo Acta Tropica vol 126 no 1 pp 11ndash18 2013

[18] C G Clark ldquoExtensive genetic diversity in Blastocystis homi-nisrdquo Molecular and Biochemical Parasitology vol 87 no 1 pp79ndash83 1997

[19] S Padukone J Mandal and S Parija ldquoSevere Blastocystissubtype 3 infection in a patient with colorectal cancerrdquo TropicalParasitology vol 7 no 2 pp 122ndash124 2017

[20] 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

[21] D Meloni P Poirier C Mantini et al ldquoMixed human intra-and inter-subtype infections with the parasite Blastocystis sprdquoParasitology International vol 61 no 4 pp 719ndash722 2012

[22] L R Ash and T C Orihel ldquoParasites a guide to laboratoryprocedures and identificationrdquo American Society of ClinicalPathologists Press vol 4 no 2 p 1988 1987

[23] S M Scicluna B Tawari and C G Clark ldquoDNA barcoding ofBlastocystisrdquo Protist vol 157 no 1 pp 77ndash85 2006

[24] T A Hall ldquoBioEdit a user-friendly biological sequence align-ment editor and analysis program for Windows 9598NTrdquoNucleic Acids Research vol 41 pp 95ndash98 1999

[25] K Tamura D Peterson N Peterson G Stecher M Nei andS Kumar ldquoMEGA5 molecular evolutionary genetics analysisusing maximum likelihood evolutionary distance and max-imum parsimony methodsrdquo Molecular Biology and Evolutionvol 28 no 10 pp 2731ndash2739 2011

[26] P Librado and J Rozas ldquoDnaSP v5 a software for comprehen-sive analysis of DNA polymorphism datardquo Bioinformatics vol25 no 11 pp 1451-1452 2009

[27] M Clement D Posada and K A Crandall ldquoTCS a computerprogram to estimate gene genealogiesrdquo Molecular Ecology vol9 no 10 pp 1657ndash1659 2000

[28] M E Ramırez-Miranda D E Jimenez-Gonzalez and M ERodrıguez-Campa ldquoSındrome de intestino irritable frecuenciay relacion filogenetica de Blastocystis sp de pacientes mexi-canosrdquo Revista de Gastroenterologıa de Mexico vol 76 no 4pp 309ndash315 2011

[29] E Rodriguez B Mateos and J C Gonzalez ldquoTransicionparasitaria a Blastocystis hominis en ninos de la zona centrodel estadodeGuerreroMexicordquoParasitologıa Latinoamericana2008

[30] R D Casero F Mongi A Sanchez and J D Ramırez ldquoBlasto-cystis and urticaria Examination of subtypes andmorphotypesin an unusual clinical manifestationrdquo Acta Tropica vol 148 pp156ndash161 2015

[31] A F Malheiros C R Stensvold C G Clark G B Bragaand J J Shaw ldquoShort report Molecular characterization ofBlastocystis obtained frommembers of the indigenous tapirapeethnic group from the Brazilian Amazon Region Brazilrdquo TheAmerican Journal of Tropical Medicine and Hygiene vol 85 no6 pp 1050ndash1053 2011

[32] J D Ramırez L V Sanchez D C Bautista A F Corredor AC Florez and C R Stensvold ldquoBlastocystis subtypes detectedin humans and animals fromColombiardquo Infection Genetics andEvolution vol 22 pp 223ndash228 2014

[33] T C Tan K G Suresh and H V Smith ldquoPhenotypic and geno-typic characterisation of Blastocystis hominis isolates implicatessubtype 3 as a subtype with pathogenic potentialrdquo ParasitologyResearch vol 104 no 1 pp 85ndash93 2008

[34] D J Stenzel and P F L Boreham ldquoBlastocystis hominisrevisitedrdquo Clinical Microbiology Reviews vol 9 no 4 pp 563ndash584 1996

[35] R AbuOdeh S Ezzedine A Samie C R Stensvold and AElBakri ldquoPrevalence and subtype distribution of Blastocystis inhealthy individuals in SharjahUnitedArabEmiratesrdquo InfectionGenetics and Evolution vol 37 pp 158ndash162 2016

[36] E B David S Guimaraes A P De Oliveira et al ldquoMolecularcharacterization of intestinal protozoa in two poor communi-ties in the State of Sao Paulo Brazilrdquo Parasites amp Vectors vol 8no 1 article no 103 2015

[37] C R Stensvold and C G Clark ldquoCurrent status of Blastocystisa personal viewrdquo Parasitology International vol 65 no 6 pp763ndash771 2016


[38] G-B Vargas-Sanchez M Romero-Valdovinos C Ramirez-Guerrero et al ldquoBlastocystis isolates frompatients with irritablebowel syndrome and from asymptomatic carriers exhibit simi-lar parasitological loads but significantly different generationtimes and genetic variability across multiple subtypesrdquo PLoSONE vol 10 no 4 pp 1ndash13 2015

[39] G Villalobos G E R Orozco-MosquedaM Lopez-Perez et alldquoSuitability of internal transcribed spacers (ITS) as markers forthe population genetic structure of Blastocystis spprdquo Parasitesamp Vectors vol 7 p 461 2014

[40] J D Ramırez A Sanchez C Hernandez et al ldquoGeographicdistribution of human Blastocystis subtypes in South AmericardquoInfection Genetics and Evolution vol 41 pp 32ndash35 2016

[41] D A Joy ldquoEarly origin and recent expansion of Plasmodiumfalciparumrdquo Science vol 300 no 5617 pp 318ndash321 2003

[42] J C Yomb S Jonckheere G Colin et al ldquoImported malariain a tertiary hospital in Belgium epidemiological and clinicalanalysisrdquo Acta clinica Belgica vol 68 no 2 pp 101ndash106 2013

[43] A P Oliveira-Arbex E B David and S Guimaraes ldquoBlasto-cystis genetic diversity among children of low-income daycarecenter in Southeastern Brazilrdquo Infection Genetics and Evolutionvol 57 pp 59ndash63 2018

[44] S H F Hagmann P V Han W M Stauffer et al ldquoTravel-associated disease among US residents visiting US GeoSentinelclinics after return from international travelrdquo Journal of FamilyPractice vol 31 no 6 pp 678ndash687 2014

[45] S Buhler R Ruegg R Steffen C Hatz and V K Jaeger ldquoAprofile of travelers - An analysis from a large Swiss travel clinicrdquoJournal of Travel Medicine vol 21 no 5 pp 324ndash331 2014

[46] H S Cheong K-T Kwon J-Y Rhee et al ldquoImported malariain Korea a 13-year experience in a single centerrdquo The KoreanJournal of Parasitology vol 47 no 3 pp 299ndash302 2009

Hindawiwwwhindawicom

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1 Introduction

Blastocystis (Heterokonta Stramenopiles) is a genus com-prising parasitic protists that inhabit the digestive tractof several metazoans such as fishes amphibians birdsreptiles rodents and humans [1ndash3] Blastocystis is globallydistributed showing a high rate of infection from underde-veloped to developed countries [4 5]

This parasite is often transmitted via the oral-fecal routeto people who work directly with animals such as thoseinvolved in intensive animal farming or industrial livestockproduction [6] In humans the signs and symptoms asso-ciated with Blastocystis infection range from diarrhea toflatulence bloating and abdominal discomfort [7 8] withthe ldquoirritable bowel syndromerdquo (IBS) being the most frequentclinical manifestation [8ndash11]

Molecular evidence based on the small subunit ribosomalRNA (SSU-rDNA) gene suggests that at least 17 geneticsubtypes can be recognized within Blastocystis [12] Nineof these subtypes are found in humans with subtype 3(ST3 hereafter) being the most common in epidemiologicalstudies worldwide [12ndash18] ST3 has been regarded to triggerIBS in humans [8] and recent research also suggests anassociation between this subtype and colorectal cancer [19]Other studies however suggest a lack of association betweenthe ST3 and some type of symptomatology in humans [7 20]While Blastocystis ST3 has medical importance and highprevalence in humans the realmagnitude of its genetic diver-sity and geographical distribution remains so far unknown[5 17] Apparently the ST3 exhibits a broader geographicaldistribution and higher genetic diversity than other geneticsubtypes of Blastocystis [16 21] but this hypothesis still needsto be tested using genetic data and clinical cases from bothwell-studied and undersampled geographical areas In thiscontext there are very few geographical and genetic data onBlastocystis ST3 fromMexico

Herein we aimed to provide newdata on the geographicaldistribution and genetic diversity of Blastocystis ST3 from arural and asymptomatic human population in Mexico Todo so we collected and targeted the SSU-rDNA region infecal samples from this population and further comparedits genetic diversity and structure with those previouslyobserved in populations of Blastocystis ST3 from otherregions of the planet

2 Materials and Methods

21 Ethical Considerations The protocol used in this studywas conducted under the ethical principles and approval ofboth the Mexican Commission on Ethics and Research ofthe Health Ministry of the State of Morelos (Comisiones deEtica y de Investigacion del Ministerio de Salud del Estado deMorelos) and the Commission on Ethics in Research of theFacultad deMedicina of theUniversidadNacional Autonomade Mexico (UNAM) (Comite de Etica de Investigacion de laFacultad de Medicina de la Universidad Nacional Autonomade Mexico) The guidelines of the committees are basedon the Mexican Official Norm (Norma Oficial Mexicana

NOM-012-SSA3-2007) which regulates the ethical principlesof every research on humans and on laboratory animalsas well as on the Declaration of Helsinki which set ethicalprinciples regarding human experimentation developed bythe World Health Organization (WHO)

Based on the abovementioned guidelines our studyonly used samples from volunteers who were respectivelyinformed about the objectives of this research the potentialrisks (if any) and the sampling procedures We obtained aninformed consent letter from all the participants

22 Sampling and Analysis Between May and November2015 fecal samples were collected from 182 volunteers (86males and 96 females) fromPuente de Ixtla in the communityof Xoxocotla State of Morelos (Mexico) ranging in agefrom 2 to 51 years old The asymptomatic status was definedaccording to the ROME III criteria Three fecal samples werecollected from each volunteer on three consecutive daysThe samples were maintained at 4∘C and transported to thelaboratory in Mexico City on the same day of collection Asubsample of each fecal sample was smeared stained with4 Lugolrsquos iodine solution and examined under a lightmicroscope at 10x and 40x magnifications [22]

23 Amplification and Sequencing of SSU-rDNA DNA wasextracted from fresh fecal samples using QIAamp DNAstool kit (QIAGEN Hilden Germany) and following themanufacturerrsquos instructions PCR protocol targeting the SSU-rDNA was conducted according to Scicluna et al [23] Inbrief we used a total mixture of 20120583l 20120583Mof primers RD5(51015840-ATC TGG TTG ATC CTG CCAG T-31015840) and BhRDr (51015840-GAG CTT TTT AAC TGC AAC AAC G-31015840) [23] as wellas 0025U of polymerase (AmpliTaq Platinum PolymeraseInvitrogen) To verify the presence of a single band andthe size of the amplified products (approximately 600 bp)the PCR products were separated by electrophoresis inagarose gel (15) in the presence of ethidium bromide visu-alized by ultraviolet transillumination and photographedThe amplification product of a 600 bp fragment of theBlastocystis SSU-rDNA was purified and sequenced using adideoxynucleotide-terminalmethod Sequencingwas carriedout in a capillary sequencer (ABI-Avant 100 University ofWashington) The sequences obtained were edited andoranalyzed with BioEdit MEGA 50 software [24 25] and adhoc scripts from Python These sequences were comparedto sequences available in GenBank employing BLAST toestablish their identityThe final sequences were deposited inGenBank under accession numbers MF539962ndashMF540015

24 Global Genetic Diversity and Haplotype Network forBlastocystis ST3 We investigated the global genetic diversity(ie LatinAmerica Europe andAsia) withinBlastocystis ST3using the novel SSU-rDNA sequences reported in the presentstudy and those previously reported within the literature Weprovided an exhaustive list of the latter sequences (119899 = 169)and sources in the Supplementary Material (available here)We investigated the following descriptive statistics of genetic


Mic

rosc

opic

anal

ysis

Bsp

Bsp + OP

OP


Negative

67

207

5



3 Results


Subt

ypes

15

75

9

ST1

ST2

ST3






4 Discussion






Eurasia 46 35 11 074010 004473 0052 minus130307ns









H1

H3H12

H7

H11

H8

H9

H18

H14

H13

H15H4

H20

H19

H17

H10

H2

H5

H6 H16

Morelos


13 samples

1 sample











5 Conclusions



Disclosure




Acknowledgments




References



















































Volume 2018

Zoology











Volume 2018

















Advances in




Enzyme Research





Volume 2018



Mic

rosc

opic

anal

ysis

Bsp

Bsp + OP

OP


Negative

67

207

5



3 Results


Subt

ypes

15

75

9

ST1

ST2

ST3






4 Discussion






Eurasia 46 35 11 074010 004473 0052 minus130307ns









H1

H3H12

H7

H11

H8

H9

H18

H14

H13

H15H4

H20

H19

H17

H10

H2

H5

H6 H16

Morelos


13 samples

1 sample











5 Conclusions



Disclosure




Acknowledgments




References



















































Volume 2018

Zoology











Volume 2018

















Advances in




Enzyme Research





Volume 2018






Eurasia 46 35 11 074010 004473 0052 minus130307ns









H1

H3H12

H7

H11

H8

H9

H18

H14

H13

H15H4

H20

H19

H17

H10

H2

H5

H6 H16

Morelos


13 samples

1 sample











5 Conclusions



Disclosure




Acknowledgments




References



















































Volume 2018

Zoology











Volume 2018

















Advances in




Enzyme Research





Volume 2018









5 Conclusions



Disclosure




Acknowledgments




References



















































Volume 2018

Zoology











Volume 2018

















Advances in




Enzyme Research





Volume 2018
















































Volume 2018

Zoology











Volume 2018

















Advances in




Enzyme Research





Volume 2018


genetic diversity and distribution of blastocystis subtype...

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