research article molecular identification and polymorphism...

Hindawi Publishing CorporationBioMed Research InternationalVolume 2013 Article ID 789326 7 pageshttpdxdoiorg1011552013789326

Research ArticleMolecular Identification and Polymorphism Determination ofCutaneous and Visceral Leishmaniasis Agents Isolated fromHuman and Animal Hosts in Iran

Homa Hajjaran1 Mehdi Mohebali12 Setareh Mamishi3

Farzaneh Vasigheh1 Mohammad Ali Oshaghi4 Saied Reza Naddaf5

Aref Teimouri1 Gholam Hossein Edrissian1 and Zabiholah Zarei6

1 Department of Medical Parasitology and Mycology School of Public Health Tehran University of Medical SciencesTehran 14155 6446 Iran

2 Center for Research of Endemic Parasites of Iran (CREPI) Tehran University of Medical Sciences Tehran Iran3 Pediatric Infections Research Center Tehran University of Medical Sciences Tehran Iran4Department of Medical Entomology and Vector Control School of Public Health Tehran University of Medical Sciences Tehran Iran5 Department of Parasitology Pasteur Institute of Iran Tehran Iran6 School of Public Health Meshkin-Shahr Research Station Tehran University of Medical Sciences Iran

Correspondence should be addressed to Homa Hajjaran hajaranhtumsacir and Mehdi Mohebali mohebalitumsacir

Received 25 April 2013 Revised 27 July 2013 Accepted 6 August 2013

Academic Editor Georgios Theodoropoulos

Copyright copy 2013 Homa Hajjaran et alThis 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

Amplification of internal transcript spacer 1 of ribosomal RNA (ITS1-RNA) gene followed by RFLP analysis and sequencing wasused to identify the causing agents of cutaneous and visceral leishmaniasis (CL and VL) in humans and animal reservoir hosts fromvarious geographical areas in Iran We also used random amplified polymorphic DNA (RAPD-PCR) to obtain polymorphismsamong isolates of Leishmania spp Totally 362 suspected human and animal cases including 173 CL 49 VL 60 rodents and 80domestic dogs were examined for Leishmania infection From 112 culture-positive samples prepared from CL cases 75 (67) wereinfected with L major and 37 (33) with L tropica Of the 60 rodents examined 25 (416) harbored the Leishmania infection21 were infected with L major and 4 with L turanica From 49 suspected VL 29 were positive by direct agglutination test (DAT)whereas microscopy detected parasite in bone marrow of 25 and culture in 28 of the patients Two VL patients were infectedwith L tropica and 26 with L infantum Of the 80 domestic dogs 56 showed anti-Leishmania antibodies with DAT Of these 55were positive by both microscopy and culture Molecular identity obtained only for 47 samples revealed L infantum in 43 andL tropica in 4 dogs The polymorphisms among L tropica and L major isolates were 36 and 73 the rate among human andcanine VL isolates was 28 and 98 respectively Our results showed that at least four different Leishmania species with variouspolymorphisms circulate among humans and animal hosts in Iran

1 Introduction

Leishmaniases are a group of diseases caused by the parasitesbelonging to the genus Leishmania The disease affects 98tropical and Mediterranean countries and 15 to 2 millioncases are estimated to occur annually worldwide with upto 350 million people at risk of infection [1] Leishmaniasespresent a wide spectrum of clinical manifestations includingcutaneous (CL) diffuse cutaneous (DCL) mucocutaneous

(MCL) and potentially fatal visceral leishmaniasis (VL) alsoknown as ldquoKala-Azarrdquo that are largely caused by differentLeishmania species In old world three species caused CL Ltropica L major (both usually cause localized skin lesions)and L aethiopica that cause nonulcerative disseminatedcutaneous lesions VL is caused by parasites of the Ldonovani-L infantum complex Recently L tropica has beenimplicated in several human cases with VL manifestations[2] In Iran the etiological agents of anthroponotic CL (ACL)

2 BioMed Research International

and zoonotic CL (ZCL) are known to be L tropica and Lmajor respectively L infantum the principal agent of VLcauses splenomegaly and hepatomegaly [3] Most of ACLare reported from northeastern and central parts of Iran [4ndash6] while ZCL extends from northeast to center and west ofIran and covers all southern provinces as well [7ndash9] Morethan 90 of the visceral cases in Iran are reported fromthe northwest and some southern provinces including Farsand Bushehr [10] All the three species of Leishmania couldbe found in some areas like Fars province in the south[11] In Iran the main reservoir hosts for ACL are humaninfected patients for ZCL are some of the desert rodentsparticularly the great gerbils (Rhombomys opimus) and forVL are domestic and wild canines (mainly domestic dogs)[4 12ndash15] In addition to the classic clinical leishmaniasismanifestations there is a variety of atypical signs in CLincluding lupoid mucosal lesions and disseminated formsand in VL including symptomatic and asymptomatic forms[16ndash18] The diagnosis of clinical forms of leishmaniasis iscommonly based on visualization of the parasite in Giemsa-stained smears bymicroscopy and culture Despite sensitivityand specificity such methods fail to identify the infectingLeishmania species [19 20] Over the last two decades DNA-based approaches including PCR followed by sequencing orRFLP analysis and species-specific PCR have been widelyused for the identification of Leishmania species [20ndash23]

Our study aimed to identify different Leishmania speciesin some foci of Iran and obtain polymorphisms among thespecies using RAPD-PCR

2 Materials and Methods

21 Sampling

211 Cutaneous Leishmaniasis Lesion aspirates wereobtained from 173 suspected CL patients referred eitherto leishmaniasis laboratory at the School of Public HealthTehran University of Medical Sciences (TUMS) or districthealth centers (DHCs) and hospitals in various regions ofIran from 2004 to 2012 Most patients were from northernnortheastern western central and southern areas of thecountry Samples were collected by injecting 02mL ofsterile saline into the dermis of the internal border of skinlesions with sterile insulin syringes After suction the samplewas transferred to RPMI-1640 medium culture and someportion was checked for Leishmania infection by microscopyfollowing the preparation of Giemsa-stained smears [22]

212 Rodents Sixty rodents including 36 Rhombomysopimus 5Meriones libycus 13 Nesokia indica 5 Tatera indicaand one Mus musculus were trapped by live baits traps fromCL endemic areas in the northeast and west of Iran None ofthe animals had any acute cutaneous lesion For preparationof samples the external edges of the ear lobes were cutwith scissors after washing and disinfection and then lowamounts of the exudates were transferred to culture media[24] Giemsa-stained smears were prepared from the samesamples

213 Visceral Leishmaniasis Blood samples were taken from49 suspected VL cases presented with fever weakness andhepatosplenomegaly at the medical health centers or leish-maniasis laboratory at the School of Public Health (SPH)TUMS Most patients were from northwestern westerncentral and southern areas of the country Also the bonemarrow aspirate was taken from iliac of the same patientsunder local anesthesia by physicians

Also blood samples were taken from eighty dogs livingin VL endemic area in the northwest and localities in thenortheast and center of the country Of the 80 dogs 59presented one or several of the typical clinical signs (such aslymphadenopathy hair loss dermal lesions onychogryposisand cachexia) and 56 had showed anti-L infantum antibod-ies All of the 59 dogswith typical clinical signswere sacrificedafter obtaining the consent of their owners and Giemsastained- smears were prepared from liver and spleen tissues

Peripheral blood samples were collected into tubes withsodium citrate anticoagulant 4 (Merck Germany) for PCRtesting and into tubes without anticoagulant for DAT Allsamples used for serology and PCRwere stored atminus20∘Cuntiluse

All experiments on the humans and animals were per-formed according to the guidelines of the Ethical Board ofTehran University of Medical Sciences Iran

214 Parasite Culture and Cryopreservation All the spec-imens including exudates from human skin lesions androdents ears aspirates from bone marrow of human patientsand liver and spleen of dogs were cultured in RPMI-1640medium (Gibco Life technologies GmbH Frankfurt Ger-many) supplemented with 10ndash15 fetal bovine serum (GibcoGermany) 100UmL penicillin and 100 ugmL streptomycin(Gibco Germany) and incubated at 24-25∘C Five days laterthe last subcultured parasites were harvested washed insterile phosphate buffered saline (PBS pH 72ndash74) and keptin minus20∘ until used All the cultures specimens were preservedin liquid nitrogen for further possible use

215 Preparation of DATAntigen and Performance of the TestL infantum (MCANIR07Moheb-gh GenBank accessionnumber FJ555210) was cultured in RPMI1640 The parasiteswere harvested at early stage of stationary phase (about 120 hlater) washed with PBS (pH = 72) and subjected to trypsindigestionTheparasites were stainedwithCoomassie brilliantblue R-250 dye (Sigma USA) fixed with formaldehyde(Merck Germany) 12 and used for DAT on humans anddogsrsquo sera DAT was performed to determine the presenceof the anti-Leishmania antibodies [14 25 26] The titers ge1 3200 and ge320 were considered positive for humans anddogs respectively [10]

216 DNA Extraction PCR and RFLP DNA was extractedfrom cultured parasites from human skin lesions rodentsrsquoears exudates and whole blood samples of suspected VLpatients and canine visceral leishmanissis dogs by using theHigh Pure PCRTemplate PreparationKit (RocheDiagnostics

BioMed Research International 3

(a) (b)

Figure 1 (a) Random amplified polymorphic DNA (RAPD) profiles obtained from Leishmania stocks and isolates with the AB1-O7 primerLane 1 L infantum stock lanes 2ndash12 L infantum isolates (b) Lane 1 L tropica stock lanes 2-3 L tropica M 100 bp size marker (Roche)

GmbH Mannheim Germany) according to the manufac-turerrsquos instructions Partial ITS1 of ribosomal RNA (ITSI-rRNA) gene of the parasites was amplified using the primersLITSR (51015840-CTGGATCATTTTCCGATG-31015840) and L58S (51015840-TGATACCACTTATCGCACTT-31015840) andPCRconditions out-lined by others [19] Amounts of 10120583L of amplicons weredigested with Fast digestion HAEIII (BsuRI) enzyme (Fer-mentas GmbH Thermo Scientific Germany) according tothe manufacturerrsquos instructions and the fragments werevisualized on 3 agarose gels [22] Leishmania species wereidentified based on obtained patterns alongside referencespecies including L tropica (GenBank accession numberEF653267) Lmajor (GenBank accession number JN860745)L turanica (GenBank accession number EU395712) and Linfantum (GenBank accession number EU810776)

217 Variation Analyses by RAPD-PCR RAPD-PCR wasperformed for 46 DNA extracted from cutaneous and vis-ceral cultured parasites samples with the primer AB1-O751015840-GGTGACGCAG-31015840 and PCR conditions outlined by others[4 27] This primer was chosen due to its reproducibility andits ability to reveal inter- and intraspecies variation

Variations among isolates of different species were calcu-lated based on profile of bands produced by AB1-O7 primer(Figure 1) A matrix for each of the isolates was constructedwith the numbers ldquo1rdquo and ldquo0rdquo corresponding to the presenceand absence of each possible band For determining thepolymorphism rate among different Leishmania species weidentified the bands that were common in less than 50of theisolates (polymorphic bands) using the following equationswhere 119885 was calculated by (1) for each polymorphic band119885-test was used for estimating the relative risk between twoindependent groups [28 29] If the obtained value for 119911in a band is less than lt164 with a confidence interval ofmore than 95 (119875 lt 005) then it will be included asa polymorphic band in the matrix After determining thepolymorphic bands polymorphism rate was calculated via(2) where 119883 is the number of isolates which contain a

polymorphic band 119873 is the total number of isolates in eachgroup and119872 is the number of total bands in each isolate

119885 =

(119883119873) minus 05

radic

(05 times 05) 119873

(1)

Polymorphism rate = 119883119872 times119873

times 100(2)

218 Nucleotide Sequence and Phylogenic Analyses Partialsequences of ITS1 gene from 20 cutaneous and visceralLeishmania isolates (Table 1) were sequenced using LITSR asthe forward primer The sequences were aligned by ClustalXsoftware the similarities among them were calculated anda phylogenic tree was constructed by using Tamura 3-parameter option of the maximum likelihood method byMEGA 5 software

3 Results

31 Cutaneous Leishmaniasis

311 Human Among 173 suspected CL patients 122 (705)were positive by microscopy and 112 (647) by culturemethod (Figure 2) DNA extracted from 112 culture positivesamples yielded an amplicon of 300ndash350 bp Digestion ofamplicons with HAEIII (BsuRI) produced two profiles 75amplicons (67) produced two bands of 220 bp and 140 bpindicative of L major and other 37 (33) produced threebands of 200 bp 60 bp and 50 bp indicative of L tropica(Figure 3) Almost all of the L tropica isolates originatedfrom northeastern areas while L major isolates belongedto the center west southern and southwest of the country(Figure 4)

312 Rodents Of the 60 caught rodents 25 (24R opimus andone N indica) were parasitologically positive (microscopyandor culture) Among the infected R opimus 21 harbored


Sample collection

Human cutaneous leishmaniasis(number 173)

Canine visceral leishmaniasis(number 80)

Leishmania species Leishmania species Leishmania species Leishmania species

Human visceral leishmaniasis(number 49)

Rodent cutaneous leishmaniasis(number 60)

Microscopy+ = 122

PCR+= 112

Culture+ = 112

Microscopy+ = 25

PCR+= 25

Culture+ = 25

BM microscopy+ = 25

B PCR+= 28

BM culture+ = 28

DAT+= 29

S and L microscopy+ = 55

B PCR+= 47

Culture+ L S = 55

DAT+= 56

L tropica = 37L major = 75

L major = 21L turanica = 4

L infantum = 26L tropica = 2


Figure 2 Flow chart which outlines the major parts of the study and results B PCR blood PCR BM bone marrow S spleen tissue L livertissue and DAT direct agglutination test

Figure 3 Restriction fragment length polymorphism (RFLP) pat-terns obtained from Leishmania stocks and patients samples lane 1negative control lane 2 L tropica lanes 3 4 L major lanes 5 6 LinfantumM 100-bp size marker (Fermentas)

L major and 3 L turanica The infecting species in N indicawas L turanica (Figure 2)

32 Visceral Leishmaniasis

321 Human From 49 suspected VL patients 29 (592)had anti-Leishmania antibodies and were considered positive(titers ge 1 3200) Microscopy and culture methods identifiedLeishmania infection in 25 (5102) and 28 (5714) individ-uals respectively ITS1-PCR using the DNA from 49 patientsrsquoblood yielded the expected band only in 28 cases (5714)Digestion of amplicons with HAEIII (BsuRI) yielded threebands of 200 bp 80 bp and 60 bp in 26 (9286) isolatesindicative ofL infantum and three bands of 200 bp 60 bp and50 bp in 2 (714) isolates indicative of L tropica (Figure 3)

322 Domestic Dogs Analyses of sera from80 domestic dogsshowed that 56 (70) were positive (at titers ge 1 320) Whole

Figure 4 Distribution of Leishmania species isolated from humanand animal hosts from some parts of Iran identified based on ITS1-PCR-RFLP (2004ndash2012)

blood dog DNA samples were successfully amplified in only47 (5875) cases RFLP analyses revealed L infantum in 43dogs (915) and L tropica in 4 dogs (85) Of the 59 dogswith typical symptomatic VL signs (including the 56 DATserologically positive cases) 55 showed infection in spleenand liver with both microscopy and culture (Figure 2)

33 RAPD-PCR and Polymorphism Rate Thepolymorphismrate among L tropica isolates was 36 The rate among Lmajor isolates was 73 and among human VL isolates was28 The highest rate (98) was found among canine VLisolates

34 Phylogenetic Tree The Leishmania isolates were groupedinto four main clads representing L major L turanica Linfantum and L tropica Within the clad L major was moreassociated with L turanica than L infantum The L tropicasupports a clear divergence between L tropica isolates from


02

9199

998693

80

90

L majorL majorL majorL majorL majorL major

(JX289855)(JX289857)(JX289859)(JX289869)(JX289868)(JX289861)

L major (JX289867)

L turanicaL turanicaL turanicaL turanica

(JN860729)(JN860733)(JN860748)(JN860758)(JX289853)(JX289852)(JX289879)

L infantumL infantumL infantumL infantum

(JX289880)L tropica (JN860722)

L tropica (JN860725)

L tropica (JX289846)L tropica (JX289848)

L tropica (JX289850)T brucei (JN673391)

Figure 5 Phylogenic tree of the ITS1 region nucleotide sequences of Leishmania isolates recovered from humans and reservoir animalsinfectedwith cutaneous and visceral leishmaniasisThe tree was constructed by using themaximum likelihood Tamura 3-parametermodel inMEGA software version 5The numbers above the branches indicate the percentage of bootstrap Branches without numbers have frequenciesof less than 70

Table 1 The accession numbers of Leishmania ITS1 sequences in Iranian species submitted in GenBank

No Code Place of isolation cityprovince Disease Source RFLPSEQ Species ACCNO1 MHOMIR10Esfahan-1 EsfahanEsfahan CL Human ++ L major JX2898552 MHOMIR10Esfahan-3 EsfahanEsfahan CL Human ++ L major JX2898573 MHOMIR10Esfahan-5 EsfahanEsfahan CL Human ++ L major JX2898594 MHOMIR10Esfahan-7 EsfahanEsfahan CL Human ++ L major JX2898615 MHOMIR05Kermanshah kr3 KermanshahKermanshah CL Human ++ L major JX2898696 MHOMIR05Kermanshah kr1 KermanshahKermanshah CL Human ++ L major JX2898677 MHOMIR05Kermanshah kr2 KermanshahKermanshah CL Human ++ L major JX2898688 MHOMIR12Bam2 BamKerman CL Human ++ L tropica JX2898469 MHOMIR12Bam4 BamKerman CL Human ++ L tropica JX28984810 MHOMIR12Bam6 BamKerman CL Human ++ L tropica JX28985011 MHOMIR02M12-Khorasan MashhadKhorasan CL Human ++ L tropica JN86072212 MHOMIR02M39-Khorasan MashhadKhorasan CL Human ++ L tropica JN86072513 MRHOIR11GOL-5 Golestan CL Rodent ++ L turanica JN86074814 MRHOIR10Khorasan6 Khorasan CL Rodent ++ L turanica JN86073315 MRHOIR11GOL-19 Golestan CL Rodent ++ L turanica JN86072916 MRHOIR11GOL-15 Golestan CL Rodent ++ L turanica JN86075817 MCANIR08Moshfe237-VL-8 MeshkinshahrArdebil VL Canine ++ L infantum JX28987918 MCANIR08Moshfe240-VL-9 MeshkinshahrArdebil VL Canine ++ L infantum JX28988019 MCANIR11Zare BomehenTehran VL Canine ++ L infantum JX28985220 MCANIR11Ziaee MeshkinshahrArdebil VL Canine ++ L infantum JX289853


the other three species The numbers above the branchesindicate the percentage of bootstrap samplings There wasno clear grouping among the 20 isolates according to theirgeographical origin (Figure 5)

Details of the specimens sequenced and submitted toGenBank are shown in Table 1

4 Discussion

In this study we could identify four species of Leishmaniaincluding L major L tropica L infantum and L turanica byITS1 gene followed by RFLP and sequencing The molecularresults on the Leishmania isolated from human cutaneouslesions are consistent with the epidemiologic studies com-mitted in recent decades [3] The results showed the majorcauses of CL in Iranwere L major (67) and L tropica (33)The major foci of ZCL transmission were in the northern(Golestan) northeastern near Turkmenistanrsquos border central(Esfahan) western (Kermanshah) near the Iraqi border andsouthern (Khuzestan) provinces of Iran The major foci ofACL transmission were in the northeast (Razavi Khorasanprovince) and center of Iran in the city of Bam (Kermanprovince) especially after the earthquake in 2003

L major was found to be the most prevalent species in Ropimus (the main reservoirs of ZCL) followed by L turanicaas the second agent [13] L turanica is incapable of infectinghumans but it causes lesions in laboratory animals [30]

In suspected VL patients DAT showed presence of anti-L infantum antibodies in 29 (592) of the human casesPCR detected Leishmania DNA in blood samples of 28(5714) individuals RFLP analyses revealed L infantum asthe main causative agent of VL (9286) and L tropica as thesecondary agent (714)This finding is in agreement with theresults of other studies performed in Iran [10 16] The mainendemic areas of VL were Ardebil province (Meshkin-Shahrdistrict) in the northwest and Fars provinces in the southHIV-Leishmania coinfection has been reported recently fromnortheastern Iran [31]

Of the 49 bone marrow aspirates from human VL 25(5102) showed amastigote forms of Leishmania sp bymicroscopy DAT and PCR were both positive in all the 25human patients

In a similar study PCR and microscopy of bone marrowaspirates were shown to be equally sensitive in patients withmicroscopically confirmed VL Moreover PCR could detectLeishmania DNA in bone marrow aspirates in 667 ofsuspectedVL patients whilemicroscopy of the samematerialwas negative [32]

Serology was more sensitive than PCR in diagnosis ofsuspected VL dogs As in humans L infantum was the maincausing agent of VL in dogs (915) and L tropica wasfound in 85 of the cases One of the important findingsof this study was molecular detection of L tropica DNA ina considerable number of humans and dogs with VL

In a similar study microscopy detected parasite in liverand spleen of 932 of symptomatic dogs while serologywas positive in 949 and PCR in 766 of cases [17] PCRseems to be less sensitive than microscopy or serology for the

detection of Leishmania in blood of suspected VL dogs [33]Phylogenic trees derived from the ITS1 sequences supporta clear divergence between L tropica from the other threespecies but there was no clear grouping among the isolatesaccording to their geographical origin In RAPD-PCR assaywe used the primer AB1-07 which had the most consistencyrate between different populations of Leishmania strains indifferent regions

5 Conclusions

Characterization of Leishmania isolates collected from dif-ferent parts of Iran showed that L tropica with 36 poly-morphisms was the primary agent in the ACL foci and Lmajorwith 73 genetic variationswas the predominant agentin the ZCL areas of Iran Moreover L infantum with 28genetic variations in human VL and 98 polymorphismsin canine VL was found in VL endemic areas of Iran Theresults of this study showed that PCR-RFLP and RAPD-PCRdespite some limitations are simple and powerful tools forthe characterization and determination of Leishmania speciespolymorphisms

Conflict of Interests

All the authors declare that they have no conflict of interests

Acknowledgments

This study received financial support from Tehran Universityof Medical Sciences (Projects nos 2403984 and 9013462)Tehran Iran The authors thank Dr B Akhoundi and Mrs SCharehdar for their collaboration in conducting the research

References

[1] ldquoControl of the Leishmaniasesrdquo Report of a Meeting of theWHO Expert Committee on the Control of LeishmaniasesGeneva switzerland 2010

[2] A J Magill M Grogl R A Gasser Jr W Sun and C N OsterldquoVisceral infection caused by Leishmania tropica in veterans ofoperation desert stormrdquo The New England Journal of Medicinevol 328 no 19 pp 1383ndash1387 1993

[3] A Nadim E Javadian M Mohebali and A Zamen MomenildquoLeishmania parasites and Leishmaniasesrdquo in Epidemiology ofLeishmaniasis in Iran Tehran University Press Tehran 2009(Persian)

[4] H Hajjaran M Mohebali M R Razavi et al ldquoIdentification ofLeishmania species isolated fromhuman cutaneous leishmania-sis using random amplified polymorphic DNA (RAPD-PCR)rdquoIranian Journal of Public Health vol 33 no 4 pp 8ndash15 2004

[5] I Sharifi S Poursmaelian M R Aflatoonian et al ldquoEmergenceof a new focus of anthroponotic cutaneous leishmaniasis dueto Leishmania tropica in rural communities of Bam districtafter the earthquake Iranrdquo Tropical Medicine and InternationalHealth vol 16 no 4 pp 510ndash513 2011

[6] M R Shiee H Hajjaran M Mohebali et al ldquoA molecular andparasitological survey on cutaneous leishmaniasis patients fromhistorical city of kashan in Isfahan province center of Iranrdquo


Asian Pacific Journal of Tropical Disease vol 2 no 6 pp 421ndash425 2012

[7] S Maraghi A Samarbaf Zadeh A A Sarlak M B Ghasemianand B Vazirianzadeh ldquoIdentification of cutaneous leishmania-sis agents by nested polymerase chain reaction (Nested-PCR)in shush city khuzestan province Iranrdquo Iranian Journal ofParasitology vol 2 no 3 pp 13ndash15 2007

[8] N Rahbarian A Mesgarian M Mahmoudi Rad et al ldquoIdenti-fication of Leishmania species isolated from human cutaneousleishmaniasis using PCRmethodrdquo Journal of Research in HealthSciences vol 9 no 2 pp 48ndash51 2009

[9] S Alimoradi H Hajjaran M Mohebali and F MansourildquoMolecular identification of Leishmania species isolated fromhuman cutaneous leishmaniasis by RAPD-PCRrdquo Iranian Jour-nal of Public Health vol 38 no 2 pp 44ndash50 2009

[10] M Mohebali ldquoEpidemiological status of visceral leishmaniasisin Iran experiences and review of literaturerdquo Journal of ClinicalExperimental Pathology vol S3 article 003 2012

[11] S Razmjou H Hejazy M H Motazedian M Baghaei MEmamy and M Kalantary ldquoA new focus of zoonotic cutaneousleishmaniasis in shiraz Iranrdquo Transactions of the Royal Societyof Tropical Medicine and Hygiene vol 103 no 7 pp 727ndash7302009

[12] H Hajjaran M Mohebali M R Abaei M R Oshaghi ZZarei et al ldquoNatural infection and phylogenetic classificationof Leishmania sppinfecting Rhombomys opimus a primaryreservoir host of zoonotic cutaneous leishmaniasis in northeastIranrdquo Transactions of the Royal Society of Tropical Medicine andHygiene vol 107 no 9 pp 550ndash557 2013

[13] A A Akhavan M R Yaghoobi-Ershadi H Mirhendi et alldquoMolecular epizootiology of rodent leishmaniasis in a hyperen-demic area of Iranrdquo Iranian Journal of Public Health vol 39 no1 pp 1ndash7 2010

[14] M Mohebali H Hajjaran Y Hamzavi et al ldquoEpidemiologicalaspects of canine visceral leishmaniosis in the Islamic Republicof Iranrdquo Veterinary Parasitology vol 129 no 3-4 pp 243ndash2512005

[15] M A Oshaghi Y Rassi L Tajedin et al ldquoMitochondrial DNAdiversity in the populations of great gerbilsRhombomys opimusthe main reservoir of cutaneous leishmaniasisrdquo Acta Tropicavol 119 no 2-3 pp 165ndash171 2011

[16] S Jafari M Hajiabdolbaghi M Mohebali H Hajjaran and HHashemian ldquoDisseminated leishmaniasis caused byLeishmaniatropica in HIV-positive patients in the Islamic Republic of IranrdquoEastern Mediterranean Health Journal vol 16 no 3 pp 340ndash343 2010

[17] A Moshfe M Mohebali G Edrissian et al ldquoCanine visceralleishmaniasis asymptomatic infected dogs as a source of Linfantum infectionrdquo Acta Tropica vol 112 no 2 pp 101ndash1052009

[18] H Hajjaran M Mohebali A A Akhavan A Taheri BBarikbin and S Nassiri ldquoUnusual presentation of disseminatedcutaneous leishmaniasis due to Leishmania major case reportsof four Iranian patientsrdquo Asian Pacific Journal of TropicalMedicine vol 6 no 4 pp 333ndash336 2013

[19] G Schonian ANasereddin NDinse et al ldquoPCRdiagnosis andcharacterization of Leishmania in local and imported clinicalsamplesrdquoDiagnosticMicrobiology and InfectiousDisease vol 47no 1 pp 349ndash358 2003

[20] M Soto R Laura M A Pineda et al ldquoSearching genesencoding Leishmania antigens for diagnosis and protectionrdquo

Scholarly Research Exchange vol 2009 Article ID 173039 25pages 2009

[21] J Alvar I D Velez C Bern et al ldquoLeishmaniasis worldwide andglobal estimates of its incidencerdquo PLoS One vol 7 no 5 ArticleID e35671 2012

[22] H Hajjaran F Vasigheh M Mohebali S Rezaei S Mamishiand S Charedar ldquoDirect diagnosis of Leishmania species onserosity materials punctured from cutaneous leishmaniasispatients using PCR-RFLPrdquo Journal of Clinical Laboratory Anal-ysis vol 25 no 1 pp 20ndash24 2011

[23] M A Oshaghi N Maleki Ravasan M Hide E Javadian YRassi et al ldquoDevelopment of species-specific PCR and PCR-restriction fragment length polymorphism assays for L infan-tum-donovani- discriminationrdquo Experimental Parasitology vol122 no 1 pp 61ndash65 2009

[24] M Mohebali E Javadian M R Yaghoobi-Ershadi A AAkhavan H Hajjaran and M R Abaei ldquoCharacterization ofLeishmania infection in rodents from endemic areas of theIslamicRepublic of IranrdquoEasternMediterraneanHealth Journalvol 10 no 4-5 pp 591ndash599 2004

[25] G H H Edrissian H Hajjaran M Mohebali G Soleiman-zadeh and S Bokaei ldquoApplication and evaluation of directagglutination test in serodiagnosis of visceral leishmaniasis inman and canine reservoirs in Iranrdquo Iranian Journal of MedicalSciences vol 21 pp 119ndash124 1996

[26] M Mohebali G H H Edrissian A Nadim H Hajjaran BAkhoundi et al ldquoApplication of direct agglutination test (DAT)for the diagnosis and seroepide-miological studies of visceralleishmaniasis in Iranrdquo Iranian Journal of Parasitology vol 1 no1 pp 15ndash25 2006

[27] S R Naddaf Dezfouli M A Oshaghi H Vatandoost EJavadian Z Telmadarei andM Assmar ldquoUse of random ampli-fied polymorphic DNA polymerase chain reaction (RAPD-PCR) and ITS2 PCR assays for differentiation of populationsand putative sibling species of Anopheles fluviatilis (DipteraCulicidae) in Iranrdquo Iranian Journal of Public Health vol 31 no3-4 pp 133ndash137 2002

[28] S A Albustan M A Alnaqeeb N Y Murad and A F Al-Alawi ldquoGenetic variation of inbred laboratory rats by RAPD-PCRrdquo Kuwait Journal of Science and Engineering vol 28 no 2pp 391ndash399 2001

[29] J M Lachin ldquoRelative risk estimates and tests for two indepen-dent groupsrdquo in Biostatistics Methods The Assesment of RelativeRisks John WilyampSons Toronto Canada 2nd edition 2000

[30] H Hajjaran M Mohebali S Alimoradi M R Abaei andG H Edrissian ldquoIsolation and characterization of pathogenicLeishmania turanica from Nesokia indica (Rodentia Muridae)by PCR-RFLP and ITS1 sequencing in Iranrdquo Transactions of theRoyal Society of Tropical Medicine and Hygiene vol 103 no 11pp 1177ndash1179 2009

[31] R Shafiei M Mohebali B Akhoundi M S Galian F Kalantaret al ldquoEmergence of co-infection of visceral leishmaniasis inHIV-positive patients in northeast Iran a preliminary studyrdquoTravel Medicine and Infection Diseases vol 13 pp S1477ndashS89392013

[32] O F Osman L Oskam E E Zijlstra et al ldquoEvaluation ofPCR for diagnosis of visceral leishmaniasisrdquo Journal of ClinicalMicrobiology vol 35 no 10 pp 2454ndash2457 1997

[33] C Margonari J A Menezes M N Rocha et al ldquoPublicknowledge about and detection of Canine visceral leishmaniasisin urban divinopolis Brazilrdquo Journal of Tropical Medicine vol2012 Article ID 429586 8 pages 2012

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Evolutionary BiologyInternational Journal of



Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014


Genetics Research International


Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational



Enzyme Research



Microbiology


and zoonotic CL (ZCL) are known to be L tropica and Lmajor respectively L infantum the principal agent of VLcauses splenomegaly and hepatomegaly [3] Most of ACLare reported from northeastern and central parts of Iran [4ndash6] while ZCL extends from northeast to center and west ofIran and covers all southern provinces as well [7ndash9] Morethan 90 of the visceral cases in Iran are reported fromthe northwest and some southern provinces including Farsand Bushehr [10] All the three species of Leishmania couldbe found in some areas like Fars province in the south[11] In Iran the main reservoir hosts for ACL are humaninfected patients for ZCL are some of the desert rodentsparticularly the great gerbils (Rhombomys opimus) and forVL are domestic and wild canines (mainly domestic dogs)[4 12ndash15] In addition to the classic clinical leishmaniasismanifestations there is a variety of atypical signs in CLincluding lupoid mucosal lesions and disseminated formsand in VL including symptomatic and asymptomatic forms[16ndash18] The diagnosis of clinical forms of leishmaniasis iscommonly based on visualization of the parasite in Giemsa-stained smears bymicroscopy and culture Despite sensitivityand specificity such methods fail to identify the infectingLeishmania species [19 20] Over the last two decades DNA-based approaches including PCR followed by sequencing orRFLP analysis and species-specific PCR have been widelyused for the identification of Leishmania species [20ndash23]

Our study aimed to identify different Leishmania speciesin some foci of Iran and obtain polymorphisms among thespecies using RAPD-PCR

2 Materials and Methods

21 Sampling

211 Cutaneous Leishmaniasis Lesion aspirates wereobtained from 173 suspected CL patients referred eitherto leishmaniasis laboratory at the School of Public HealthTehran University of Medical Sciences (TUMS) or districthealth centers (DHCs) and hospitals in various regions ofIran from 2004 to 2012 Most patients were from northernnortheastern western central and southern areas of thecountry Samples were collected by injecting 02mL ofsterile saline into the dermis of the internal border of skinlesions with sterile insulin syringes After suction the samplewas transferred to RPMI-1640 medium culture and someportion was checked for Leishmania infection by microscopyfollowing the preparation of Giemsa-stained smears [22]

212 Rodents Sixty rodents including 36 Rhombomysopimus 5Meriones libycus 13 Nesokia indica 5 Tatera indicaand one Mus musculus were trapped by live baits traps fromCL endemic areas in the northeast and west of Iran None ofthe animals had any acute cutaneous lesion For preparationof samples the external edges of the ear lobes were cutwith scissors after washing and disinfection and then lowamounts of the exudates were transferred to culture media[24] Giemsa-stained smears were prepared from the samesamples

213 Visceral Leishmaniasis Blood samples were taken from49 suspected VL cases presented with fever weakness andhepatosplenomegaly at the medical health centers or leish-maniasis laboratory at the School of Public Health (SPH)TUMS Most patients were from northwestern westerncentral and southern areas of the country Also the bonemarrow aspirate was taken from iliac of the same patientsunder local anesthesia by physicians

Also blood samples were taken from eighty dogs livingin VL endemic area in the northwest and localities in thenortheast and center of the country Of the 80 dogs 59presented one or several of the typical clinical signs (such aslymphadenopathy hair loss dermal lesions onychogryposisand cachexia) and 56 had showed anti-L infantum antibod-ies All of the 59 dogswith typical clinical signswere sacrificedafter obtaining the consent of their owners and Giemsastained- smears were prepared from liver and spleen tissues

Peripheral blood samples were collected into tubes withsodium citrate anticoagulant 4 (Merck Germany) for PCRtesting and into tubes without anticoagulant for DAT Allsamples used for serology and PCRwere stored atminus20∘Cuntiluse

All experiments on the humans and animals were per-formed according to the guidelines of the Ethical Board ofTehran University of Medical Sciences Iran

214 Parasite Culture and Cryopreservation All the spec-imens including exudates from human skin lesions androdents ears aspirates from bone marrow of human patientsand liver and spleen of dogs were cultured in RPMI-1640medium (Gibco Life technologies GmbH Frankfurt Ger-many) supplemented with 10ndash15 fetal bovine serum (GibcoGermany) 100UmL penicillin and 100 ugmL streptomycin(Gibco Germany) and incubated at 24-25∘C Five days laterthe last subcultured parasites were harvested washed insterile phosphate buffered saline (PBS pH 72ndash74) and keptin minus20∘ until used All the cultures specimens were preservedin liquid nitrogen for further possible use

215 Preparation of DATAntigen and Performance of the TestL infantum (MCANIR07Moheb-gh GenBank accessionnumber FJ555210) was cultured in RPMI1640 The parasiteswere harvested at early stage of stationary phase (about 120 hlater) washed with PBS (pH = 72) and subjected to trypsindigestionTheparasites were stainedwithCoomassie brilliantblue R-250 dye (Sigma USA) fixed with formaldehyde(Merck Germany) 12 and used for DAT on humans anddogsrsquo sera DAT was performed to determine the presenceof the anti-Leishmania antibodies [14 25 26] The titers ge1 3200 and ge320 were considered positive for humans anddogs respectively [10]

216 DNA Extraction PCR and RFLP DNA was extractedfrom cultured parasites from human skin lesions rodentsrsquoears exudates and whole blood samples of suspected VLpatients and canine visceral leishmanissis dogs by using theHigh Pure PCRTemplate PreparationKit (RocheDiagnostics


(a) (b)






119885 =

(119883119873) minus 05

radic

(05 times 05) 119873

(1)


times 100(2)


3 Results





Sample collection






Microscopy+ = 122

PCR+= 112

Culture+ = 112

Microscopy+ = 25

PCR+= 25

Culture+ = 25

BM microscopy+ = 25

B PCR+= 28

BM culture+ = 28

DAT+= 29


B PCR+= 47

Culture+ L S = 55

DAT+= 56
















02

9199

998693

80

90


(JX289855)(JX289857)(JX289859)(JX289869)(JX289868)(JX289861)

L major (JX289867)


(JN860729)(JN860733)(JN860748)(JN860758)(JX289853)(JX289852)(JX289879)












4 Discussion









5 Conclusions




Acknowledgments


References












































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


(a) (b)






119885 =

(119883119873) minus 05

radic

(05 times 05) 119873

(1)


times 100(2)


3 Results





Sample collection






Microscopy+ = 122

PCR+= 112

Culture+ = 112

Microscopy+ = 25

PCR+= 25

Culture+ = 25

BM microscopy+ = 25

B PCR+= 28

BM culture+ = 28

DAT+= 29


B PCR+= 47

Culture+ L S = 55

DAT+= 56
















02

9199

998693

80

90


(JX289855)(JX289857)(JX289859)(JX289869)(JX289868)(JX289861)

L major (JX289867)


(JN860729)(JN860733)(JN860748)(JN860758)(JX289853)(JX289852)(JX289879)












4 Discussion









5 Conclusions




Acknowledgments


References












































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


Sample collection






Microscopy+ = 122

PCR+= 112

Culture+ = 112

Microscopy+ = 25

PCR+= 25

Culture+ = 25

BM microscopy+ = 25

B PCR+= 28

BM culture+ = 28

DAT+= 29


B PCR+= 47

Culture+ L S = 55

DAT+= 56
















02

9199

998693

80

90


(JX289855)(JX289857)(JX289859)(JX289869)(JX289868)(JX289861)

L major (JX289867)


(JN860729)(JN860733)(JN860748)(JN860758)(JX289853)(JX289852)(JX289879)












4 Discussion









5 Conclusions




Acknowledgments


References












































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


02

9199

998693

80

90


(JX289855)(JX289857)(JX289859)(JX289869)(JX289868)(JX289861)

L major (JX289867)


(JN860729)(JN860733)(JN860748)(JN860758)(JX289853)(JX289852)(JX289879)












4 Discussion









5 Conclusions




Acknowledgments


References












































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology




4 Discussion









5 Conclusions




Acknowledgments


References












































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology






































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

research article molecular identification and polymorphism...

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