55 (2006) 277ndash283wwwelseviercomlocateparint

Parasitology International

Application of kDNA as a molecular marker to analyseLeishmania infantum diversity in Portugal

Sofia Cortes a Isabel Mauricio b Ana Almeida a Joseacute Manuel Cristovatildeo a Francine Pratlong cJean Pierre Dedet c Lenea Campino a

a Unidade de Leishmanioses Centro Malaacuteria Outras Doenccedilas Tropicais Instituto de Higiene e Medicina TropicalUniversidade Nova de Lisboa Rua da Junqueira 96 1349-008 Lisboa Portugal

b Pathogen Molecular Biology Unit Department of Infectious and Tropical Diseases London School of Hygiene and Tropical MedicineKeppel Street London WC1E 7HT UK

c Centre National de Reacutefeacuterence des Leishmania Universiteacute de Montpellier 163 rue Auguste Broussonet 34090 Montpellier France

Received 27 December 2005 received in revised form 11 July 2006 accepted 19 July 2006Available online 7 September 2006

Abstract

Around the Mediterranean basin Leishmania infantum is an important parasite causing canine leishmaniasis and visceral and cutaneous clinicalforms in both immunocompetent and immunocompromised humans Efficient monitoring and evaluation of epidemiology with discriminatorymolecular markers are required We investigated the genetic diversity of L infantum in Portugal by polymerase chain amplification and restrictionfragment length polymorphism analysis of kinetoplastid DNA as molecular marker We analysed 120 Portuguese isolates of L infantum plus 16other non-Portuguese isolates (as a reference group) from humans dogs and sand flies The Portuguese population showed a high degree ofpolymorphism with a total of 13 profiles identified The predominant profile was A which was only detected in the Portuguese samples

The kinetoplastid DNA PCR-RFLP assay described here was suitable for use directly with biological samples and the profiles obtained werestable during long-term growth in vitro and in laboratory animalscopy 2006 Elsevier Ireland Ltd All rights reserved

Keywords Leishmania infantum Epidemiology Portugal PCR-RFLP

Table 1Number and geographic distribution of Portuguese isolates from different hosts

Regions Samples

Humans Dogs Sand files Total

VL CL

ADR 8 4 3 2a 17

1 Introduction

In the Mediterranean countries Leishmania infantum isconsidered the main etiological agent of visceral leishmaniasisand one of the agents responsible for cutaneous leishmaniasis[1] The dog is considered the domestic reservoir of this parasiteplaying an important role in the transmission of the diseasethrough the vector of the genus Phlebotomus L infantum hasalso been shown to be an important opportunistic parasite in HIVinfected patients [23]

For epidemiological studies and analysis of species diversityisoenzyme analysis has been the most commonly used method

Abbreviations PCR polymerase chain reaction kDNA kinetoplast DNARFLP restriction fragment length polymorphism bp base pairs TAE Tris-acetate EDTA Corresponding author Tel +351 21 3652600 fax +351 21 3632105E-mail address campinoihmtunlpt (L Campino)

1383-5769$ - see front matter copy 2006 Elsevier Ireland Ltd All rights reserveddoi101016jparint200607003

and also a useful taxonomic tool However it has some limitationsbecause of the need for parasite cultivation and due to un-derestimation of the parasite genetic diversity nucleotide oreven amino acid substitutions that do not affect isoenzyme

LR 42 2 45 0 89ALR 1 7 0 8AR 3 1 1 1b 6Total 61 56 3 120

ADR Alto Douro region LR Lisboametropolitan region ALR Alentejo regionAR Algarve region VL visceral leishmaniasis CL cutaneous leishmaniasisaP ariasi bP perniciosus

Table 2Strains used as reference group

Species WHO code Zymodeme Geographic origin

L infantum MHOMES93PM1 1 SpainL infantum MHOMES86BCN16 1 SpainL infantum MCANES2001LLM-1007 1 SpainL infantum MCANES2002LLM-1139 1 SpainL infantum MHOMES2001LLM-981 1 SpainL infantum MHOMFR78LEM75 1 FranceL infantum MHOMFR97LSL29 1 FranceL infantum MHOMFR80LEM189 11 FranceL infantum MHOMMT85BUCK 78 MaltaL infantum MHOMBR74PP75 1 BrazilL infantum MHOMBR7246 1 BrazilL infantum MHOMSD623S 81 SudanL donovani MHOMSD82GILANI 30 SudanL donovani MHOMET00HUSSEN 31 EthiopiaL donovani MHOMET67HU3 (LV9) 18 EthiopiaL donovani MHOMET72GEBRE 1 82 Ethiopia

GILANI strain was previously classified as L infantum and GEBRE 1 as Larchibaldi

Fig 1 RFLP fragments of kDNA-PCR products from 12 DNA L infantumPortuguese samples with the enzyme BglIImdashPattern I lanes 7 9 11 pattern II1ndash6 8 10 12 250 bp molecular size marker lanes 1ndash5 canine samples lane 6sand fly sample lanes 7ndash12 human samples (7 8 immunocompromisedpatients 9ndash12 immunocompetent patients)

278 S Cortes et al Parasitology International 55 (2006) 277ndash283

electrophoretic mobility will not be detected This typing methodshould therefore be complemented with polymerase chainreaction (PCR)-basedmethods that use polymorphic DNA targetswith high discriminatory power [4] Several nuclear DNAmarkers have been used such as the ribosomal internaltranscribed spacer regions and the mini-exon [5ndash7] small sub-unit rRNA genes [8] antigen genes [4] microsatellites [9] andextranuclear DNA such as minicircles of kinetoplast DNA(kDNA) [1011] kDNA contains around 10000 minicircles percell each of around 800 base pairs (bp) in size with anapproximately 200-bp conserved region and an approximately600-bp variable region The heterogeneity of the variable regionhas been exploited to discriminate between strains of the samespecies [12] Moreover schizodeme analysis has revealed thatclosely related strains can have variable minicircle classfrequencies conferring diversity [13] Minicircle DNA isessential for the trypanosomatidsmitochondrial genetic functionas minicircles code for guide RNA which plays an essential rolein editing mRNA from maxicircles which contain genes foressential mitochondrial proteins [13]

In Portugal leishmaniasis is present throughout the countryboth in rural and urban areas Three endemic foci have beenidentified Alto Douro region Lisboa Metropolitan region andAlgarve region [14ndash16] Leishmaniasis is also present in theAlentejo Region but with low endemicity [17] L infantumzymodeme MON-1 has been isolated from 967 of all theinfection cases studied by us so far [18] In the present study we

Table 3Patterns obtained with 9 restriction enzymes and correspondent fragment size

Enzymes BglII Bme1390 I DdeI HpaII

Patterns I II I II III I II III I

Fragments (bp) 447 258 411 288 447 319 419 240 410189 36 123 100 28 180 37

36 28 28

have used kDNA-PCR amplification and further restrictionfragment length polymorphism (RFLP) analysis to examine thegenetic diversity of L infantum isolated from humans dogs andsand flies from several geographic areas of the country

2 Materials and methods

21 Isolates

A total of 136 isolates were analysed One hundred andtwenty samples were Portuguese of which 61 were obtainedfrom human biological material from bone marrow in visceralleishmaniasis cases (54) or from skin in cutaneous leishmaniasiscases (7) from 16 immunocompetent and 45 immunocompro-mised patients 56 samples were from dogs and 3 were from thevector (Phlebotomus perniciosus and P ariasi) The sampleswere obtained from the regions of Alto Douro (AD) LisboaMetropolitan Region (LR) Alentejo and Algarve (Table 1)Vector and dog samples were obtained from epidemiologicalsurveys and human samples were sent to us by pathologists andclinicians from local hospitals for diagnostics With the ex-ception of 4 strains that had been typed by isoenzyme analysis aszymodeme MON-24 (1) from LR and MON-29 (3) fromAlgarve and 7 not typed (4 from AD 2 from LR and 1 fromAlentejo) all the others belonged to zymodeme MON-1This study included 9 strains from other countries of the

RsaI VspI PstI SfcI XapI

II III IV I II IV I II I I I

287 447 350 253 210 253 161 170 297 224 348123 57 194 197 144 152 130 149 153 6037 37 40 48 134 90 70 40

50

Table 4Restriction genotypes obtained from isolates

Profiles Patterns with restriction enzymes

BglII Bme1390I DdeI HpaII RsaI VspI

A II I II I I IB I I I I I IC II I III I I ID I II I II I IE I I II I I IF I I I IV I IG I I I I II IIH II I I I II III II I I I I IIJ I I I I IV IK I III I III I IL I II I II IV IM I III I IV II IN I I I III I IO I I I I I II

Fig 2 Distribution of the 13 genotypes in the studied regions of Portugal andnumber of isolates in brackets MON-29 MON-24 diams 1 isolate not typeddiamsdiams 2 isolates not typed

279S Cortes et al Parasitology International 55 (2006) 277ndash283

Mediterranean Basin 5 East African strains and 2 Brazilian Linfantum strains (Table 2) To test stability of the marker westudied an additional group of 4 sets of two samples derived froman original isolate after long-term culture in vitro or in vivo(experimental infection of rodents and dogs)

22 DNA extraction and PCR amplification

DNA was extracted from cultured parasites or directly fromclinical samples (bone marrow or skin) using a commercialextraction kit (High Template DNA Preparation Kit RocheGermany) according to the manufacturers instructions PCRamplifications were performed in volumes of 50 μl with MC1and MC2 kinetoplastid primers specific for the Leishmaniadonovani complex [19] that amplify a partial kDNA sequenceof 447 bp To confirm the PCR amplification products weresubjected to electrophoresis in 15 agarose gel with 05 μgmlethidium bromide in 1times TAE buffer

23 Restriction fragment length polymorphism (RFLP)

For endonuclease digestion 9 restriction enzymes with 4 to6 bp restriction sites were used BglII Bme1390I DdeI HpaIIPstI RsaI SfcI VspI and XapI Endonuclease digestion wasdone with approximately 100 ng of PCR products in a total of10 μl with 8 U of restriction enzyme in the recommendedbuffer and 005 μgml bovine serum albumin at +37 degC over-night Restriction fragments were separated in 3 agarose gelwith 05 μgml ethidium bromide for 25 h The gel was thenphotographed using an Eagle Eye II closed circuit devicecamera system

24 Data analysis

Each pattern of digested kDNA-PCR product fragments thatwere analysed for all nine restriction enzymes contributed to aprofile or genotype interpreted in a qualitative analysis

The presence or absence of bands from the RFLP data wasscored (1 for presence and 0 for absence) A distance matrix wasproduced using RESTDIST from which a neighbor-joiningdendrogram was built using NEIGHBOR and a maximumlikelihood tree was produced using RESTML (both in thepackage PHYLIP version 36 available at httpevolutiongeneticswashingtoneduphyliphtml) The resulting tree wasplotted with TREEVIEW program version 16

Statistical analysis was performed using the Chi-square Testfrom the package SPSS version 130 A 5 level of significancewas used

3 Results

kDNA endonuclease digestion with the 9 restriction enzymesproduced several patterns with fromone to four visible bands in all

Table 4Restriction genotypes obtained from isolates

Profiles Patterns with restriction enzymes

BglII Bme1390I DdeI HpaII RsaI VspI

A II I II I I IB I I I I I IC II I III I I ID I II I II I IE I I II I I IF I I I IV I IG I I I I II IIH II I I I II III II I I I I IIJ I I I I IV IK I III I III I IL I II I II IV IM I III I IV II IN I I I III I IO I I I I I II

Fig 2 Distribution of the 13 genotypes in the studied regions of Portugal andnumber of isolates in brackets MON-29 MON-24 diams 1 isolate not typeddiamsdiams 2 isolates not typed

279S Cortes et al Parasitology International 55 (2006) 277ndash283

Mediterranean Basin 5 East African strains and 2 Brazilian Linfantum strains (Table 2) To test stability of the marker westudied an additional group of 4 sets of two samples derived froman original isolate after long-term culture in vitro or in vivo(experimental infection of rodents and dogs)

22 DNA extraction and PCR amplification

DNA was extracted from cultured parasites or directly fromclinical samples (bone marrow or skin) using a commercialextraction kit (High Template DNA Preparation Kit RocheGermany) according to the manufacturers instructions PCRamplifications were performed in volumes of 50 μl with MC1and MC2 kinetoplastid primers specific for the Leishmaniadonovani complex [19] that amplify a partial kDNA sequenceof 447 bp To confirm the PCR amplification products weresubjected to electrophoresis in 15 agarose gel with 05 μgmlethidium bromide in 1times TAE buffer

23 Restriction fragment length polymorphism (RFLP)

For endonuclease digestion 9 restriction enzymes with 4 to6 bp restriction sites were used BglII Bme1390I DdeI HpaIIPstI RsaI SfcI VspI and XapI Endonuclease digestion wasdone with approximately 100 ng of PCR products in a total of10 μl with 8 U of restriction enzyme in the recommendedbuffer and 005 μgml bovine serum albumin at +37 degC over-night Restriction fragments were separated in 3 agarose gelwith 05 μgml ethidium bromide for 25 h The gel was thenphotographed using an Eagle Eye II closed circuit devicecamera system

24 Data analysis

Each pattern of digested kDNA-PCR product fragments thatwere analysed for all nine restriction enzymes contributed to aprofile or genotype interpreted in a qualitative analysis

The presence or absence of bands from the RFLP data wasscored (1 for presence and 0 for absence) A distance matrix wasproduced using RESTDIST from which a neighbor-joiningdendrogram was built using NEIGHBOR and a maximumlikelihood tree was produced using RESTML (both in thepackage PHYLIP version 36 available at httpevolutiongeneticswashingtoneduphyliphtml) The resulting tree wasplotted with TREEVIEW program version 16

Statistical analysis was performed using the Chi-square Testfrom the package SPSS version 130 A 5 level of significancewas used

3 Results

kDNA endonuclease digestion with the 9 restriction enzymesproduced several patterns with fromone to four visible bands in all

Table 6Distribution of genotypes in Portuguese human isolates according to immunestatus of the patients

Genotypes Genotypes inimmunocompetent (n=16)

Genotypes inimmunocompromised (n=45)

A 4 (3 VL 1 CL) 27 (26 VL 1 CL)B 7 (5 VL 2 CL) 8 (8 VL)C 1 (1 VL) 1 (1 VL)D 1 (1 CL) 1 (1 VL)E 0 3 (3 VL)F 0 2 (2 VL)G 0 1 (1 VL)H 1 (1 CL) 0I 1 (1 VL) 0K 1 (1 CL) 0L 0 2 (2 VL)

Numbers in brackets are the number of isolates of each clinic manifestation VLvisceral leishmaniasis CL cutaneous leishmaniasis

280 S Cortes et al Parasitology International 55 (2006) 277ndash283

the 136 samples analysed Table 3 shows the pattern types (I to IV)obtained with each restriction enzyme Some patterns differed bypresenceabsence of restriction sites (eg IIIIII of HpaII) whilstothers had the same number of bands but with different sizes (egIIIV of RsaI) Restriction patterns obtained with PstI SfcI andXapI were monomorphic whereas patterns obtained with BglII(Fig 1)Bme1390IDdeIHpaIIRsaI andVspI were polymorphicThese last six enzymes were considered for genotyping

To increase the discriminatory power of the method a com-bined analysis was performed for the kDNA restriction productsobtained with each of the six polymorphic enzymes Fifteenprofilesgenotypes were established and are represented by letters(AndashO) and diversity was analysed by the number of differentgenotypes obtained (Table 4) A PCR product from each differentgenotype was sequenced confirming the presence of all therestriction sites (data not shown)

The Portuguese population showed a high degree ofpolymorphism with a total of 13 profiles identified (from A toM) and a predominance of profile A present in 60 of the 120isolates (500) This profile was exclusive to the Portuguesesamples Profile B was also common and identified in 36120(300) of the isolates whereas the frequency of the other 11profiles ranged from 58 (7120) to 08 (1120) The 89 Linfantum isolates (humans and dogs) from the LR had 9different profiles (Fig 2) with 4244 human samples isolatedfrom immunocompromised patients all with visceral leishman-iasis In the AD region 5 profiles were identified (A B D I K)in a total of 17 isolates studied of which 12 were isolated fromimmunocompetent patients 4 with cutaneous and 8 with vis-ceral leishmaniasis 3 from dogs and 2 from phlebotomine sandflies The eight samples (humans and dogs) from the Alentejoregion were classified into two profiles (A C) and the 6 samples(humans dogs and sand fly) from the Algarve region wereclassified into 4 restriction profiles (B E H J)

Table 5Genotypes and zymodemes found according to geographic origin

Geographic origin Genotypes Zymodemes

Portugal A MON-1 (57)B MON-1 (35)C MON-1 (1)D MON-1 (4)E MON-1 (7)F MON-29 (2)G MON-24 (1)H MON-29 (1)I MON-1 (1)J MON-1 (1)L MON-1 (2)M MON-1 (1)

Spain B MON-1 (3)N MON-1 (2)

France F MON-11 (1)K MON-1 (2)

Malta O MON-78 (1)Ethiopia G MON-31 (1) MON-82 (1) MON-18 (1)Sudan G MON-30 (1) MON-81 (1)Brazil B MON-1 (2)

Numbers in brackets are the number of isolates

The distribution of profiles according to zymodeme acrossthe whole sample set was as follows within zymodeme MON-1 group 11 profiles were found with a predominance of A and Bprofiles genotype H was observed in one MON-29 and F in twoother MON-29 Portuguese strains and in one MON-11 Frenchstrain A Portuguese isolate that had not been typed isoenzy-matically and twoMON-1 French strains presented genotype Kgenotypes N and O were only found in two MON-1 Spanishstrains and one MON-78 Maltese strain respectively The EastAfrican strains (zymodemes MON-30 MON-31 MON-81MON-82 and MON-18) all had the same genotype (G) as wellas the Portuguese isolate MON-24 The two Brazilian Linfantum strains typed as MON-1 presented profile B one ofthe most frequent profiles of the Portuguese isolates (Table 5)

The three MON-1 Portuguese strains isolated from sand flieshad 3 different genotypes J isolated from P perniciosus and Band A isolated from P ariasi

Both immunocompetent and immunocompromised patientspresented a diversity of profiles (Table 6) Seven profiles werefound in 16 L infantum isolates from immunocompetent patientsand 8 profiles in 45 L infantum isolates from immunocompro-mised patients The two main genotypes A and B were sig-nificantly associated with the immune status of the patientrespectively with immunocompromised and immunocompetentpatients p=0024

From 12 immunocompromised patients more than one samplewas obtained within different time intervals from 1 month to2 years corresponding to relapses or re-infections All sampleswere typed as MON-1 but in one of these patients the restrictionprofile was different between the first and the second isolate Thestability of the kDNA PCR-RFLP profiles was tested on 4different pairs of samples after long periods in vitro and afterpassage through animals No differences were detected in therestriction patterns between the original isolates and after long-term passages in vitro and in vivowith the exception of one isolatethat had a different restriction profile after more than 5 years in invitro culture The distinct genotype was due to differences in therestriction patterns of 2 enzymes

The neighbor-joining dendrogram (Fig 3) obtained from thegenetic distances derived from the kDNA PCR-RFLP data shows

Fig 3 Neighbor-joining tree using genetic distances derived from the kDNAPCR-RFLP data (genotypes) of all strains In black is the outgroup Close toeach genotype is the number of human (H) canine (C) and sand fly (S) isolatesGenotype G served as outgroup and included all African strains and aPortuguese MON-24 isolate ⋆ P ariasi P perniciosus

281S Cortes et al Parasitology International 55 (2006) 277ndash283

the relationships between the 15 genotypes Unfortunately boot-strapping was not appropriate and there is no measure of supportfor the groups found However the maximum likelihood tree (Lnlikelihood minus26463) was very similar with the exception of thecluster with genotypes D F K L M and N which had a differenttopology and also included genotype JWe have selected genotypeG as the outgroup given that it is present almost exclusively in Ldonovani strains from East Africa A Portuguese MON-24 wasalso found to have genotype G Genotype H is the closest to theroot and was found in a Portuguese MON-29 strain followed bygenotype O found in a MON-78 Maltese dermotropic strain Twomain clusters were detected with genotype B (one of the mostcommon in Portugal but present in Spain and Brazil) as a possiblecommon ancestor The cluster with genotypes E A C and I form avery clear line of descent from genotype B and have here onlybeen identified in Portugal

4 Discussion

kDNA has been exploited for diagnosis [2021] identifica-tion and genotyping of Leishmania species [10122223] In thepresent study the genetic diversity of Portuguese L infantumisolates has been evaluated using PCR-RFLP of kDNA Onehundred and twenty Portuguese isolates from humans dogs andsand flies from different regions of Portugal were analysed

The high number (13) of restriction profiles observed withinthe Portuguese samples indicates genetic heterogeneity withinthis parasite population Recent studies using different molecularmarkers and PCR-RFLP methods have uncovered genetic diver-sity within differentLeishmania species Cupolillo et al [6] usingRFLP analysis have found genetic diversity in Leishmaniabraziliensis from several regions of Brazil and most genotypeswere associated with specific geographic areas However in ourresults no genotype was exclusive to one focus perhaps because

our country is a small geographic region with migration betweenfoci

Two main genotypes (A and B) could be associated with theimmune status of the patients genotype A correlated with HIV+immunocompromised patients and B with immunocompetentpatients It is important to note that most of the LR isolates werefrom HIV+ drug addicts living in ghetto-like closed commu-nities inside Lisboa This could suggest that the population ofstrains circulating in AIDS patients may have suffered a foundereffect with propagation of a few clones This hypothesis willhave to be supported with a broader study

In the context of our total sample set within zymodemeMON-1 high diversity was observed in the sequence of kDNAanalysed as 11 restriction profiles were found Genetic diversitywithin MON-1 was also observed by others [24] In this studygenotype A the most frequent genotype was exclusive to thePortuguese population

The restriction profile was the same in all East African isolates(profile G) although they have been considered to belong todifferent zymodemes and species (L infantum L archibaldi andL donovani) [25] This finding corroborates other studies thatclassify the African strains as L donovani [2627] The presentstudy also shows the presence of one of the most frequent Portu-guese profiles (B) in the Brazilian samples Although just twoisolates have been studied this result reinforces the evidence thatL infantum and the New World parasite Leishmania chagasi areindeed synonymous species [28]

In this study we found that strains considered dermotropichad closely related genotypes (H and O) or the same genotype(G) as the outgroup strains Viscerotropic strains were heremonophyletic This is a similar result to that of Angelici et al[29] who showed that dermotropic L infantum strains weregenetically divergent from viscerotropic strains in a study ofkDNA-RFLP of 29 strains The Portuguese MON-24 strain withgenotype G (as present in the African L donovani strains) wasisolated from an intravenous drug user with AIDS It is possiblethat this strain was imported and that the kDNA genotype wasacquired by genetic exchange Such mosaic genomes have beenreported in the L donovani complex [30]

We have identified a line of descent of Portuguese geno-types formed by genotypes E A C and I in order fromgenotype B in which genotype Awas the most frequent one inthe Portuguese population It is possible that these genotypeshave evolved in Portugal The second cluster is much lessdefined and includes strains from other countries It is possiblethat these genotypes have evolved outside of Portugal and werethen imported The phylogenetic position of genotype J was notconsistent between the two tree building methods used Thisgenotype is the most distant from any other in this study and it islikely that the lack of intermediate genotypes complicates thephylogenetic analysis

In this study we assessed kDNA PCR-RFLP profile stabilitywith 4 sets of original isolates and after prolonged culture invitro after passage in vivo and with sequential isolates frompatients with several episodes of leishmaniasis Three of thestudied sets proved to be stable In one set we found a differentrestriction profile between the original isolate and after long-

282 S Cortes et al Parasitology International 55 (2006) 277ndash283

term in vitro culture indicating that the strain may have sufferedmutations in the kDNA sequences or changes in minicircle classfrequencies The change could be a consequence of adaptationdue to the pressure of long-time in vitro culture or due to agenetic driftfounder effect by sub-culturing Some authors haveproved that the mitochondrial genome can change in terms ofminicircle class frequencies due to in vitro drug exposure [31]On the other hand the results obtained on DNA of strainsisolated from patients that have had several episodes of leish-maniasis and revealed the same restriction profiles (in 11 of 12patients) confirms considerable stability among the minicirclesfrom sequential isolates before and after treatment The differentprofile observed in one case could correspond to a re-infectionrather than a relapse as was also suggested byMorales et al [32]However we cannot discard the possibility of having infectionsby mixed populations (or parasites with different frequencies ofminicircles) and a less dominant genotype becoming dominantafter treatment

We have found in the 3 phlebotomine isolates studied 3different profiles The parasites had been isolated from the twovector species (P ariasi and P perniciosus) in the country Twoof these genotypes (A B) were also identified from the samefocus in canine and human hosts According to Cupolillo et al[6] the presence of the same genotypes in humans and dogs inthe same area suggests that transmission cycles could be definedusing these methodologies

Thiswork also showed that the kDNAPCR-RFLPmethod canbe applied to parasite cultures or directly on biological samples

The results presented in this paper suggest that Portugal asmall country of South-eastern Europe considered homogenousfor the Leishmania species and zymodeme responsible forleishmaniasis presents more genetic diversity than previouslythought Further studies on L infantum diversity should becarried out with other genetic markers and analysed in conjunc-tion with our findings to reach firmer conclusions on the epi-demiology of leishmaniases in Portugal

Acknowledgments

We thank R Brazil (Centro de Pesquisas Reneacute Rachou Brazil)and C Chicharro (Instituto de Salud Carlos IIIMadrid Spain) forproviding us some of the strainsWe aremost grateful to cliniciansand pathologists from local hospitals that provided us with theclinical samples We would like to thank the European Com-mission (Contract no QLK2-CT-2001-01810) who supportedthis work and to all partners of the project with special regard tothe coordinator Michael Miles

References

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[2] Alvar J Leishmaniasis and AIDS co-infection the Spanish exampleParasitol Today 199410160ndash3

[3] Campino L Santos-Gomes G Pratlong F Dedet JP HIV-Leishmania co-infection in Portugal isolation of Leishmania infantum MON-24 Trans RSoc Trop Med Hyg 199488394

[4] Tintaya KW Ying X Dedet JP Rijal S De Bolle X Dujardin JC Antigengenes for molecular epidemiology of leishmaniasis polymorphism of

cysteine proteinase B and surface metalloprotease glycoprotein 63 in theLeishmania donovani complex J Infect Dis 20041891035ndash43

[5] El Tai N El Fari M Mauricio IL Miles MA Oskam L El Safi SH et alLeishmania donovani intraspecific polymorphisms of Sudanese isolatesrevealed by PCR-based analyses and DNA sequencing Exp Parasitol20019735ndash44

[6] Cupolillo E Brahim LR Toaldo CB Oliveira-Neto MP Brito MEFalqueto A et al Genetic polymorphism and molecular epidemiology ofLeishmania (Viannia) braziliensis from different hosts and geographicareas in Brazil J Clin Microbiol 2003413126ndash32

[7] Mauricio IL Stothard JR Miles MA Leishmania donovani complexgenotyping with the ribosomal internal transcribed spacer and the mini-exon Parasitol 2004128263ndash7

[8] van Eys GJ Schoone GJ Kroon N Ebeling SB Sequence analysis of smallsubunit ribosomal RNA genes and its use for detection and identification ofLeishmania parasites Mol Biochem Parasitol 199251133ndash42

[9] Bulle B Millon L Bart JM Gallego M Gambarelli F Portus M et alPractical approach for typing strains ofLeishmania infantumbymicrosatelliteanalysis J Clin Microbiol 2002403391ndash7

[10] Berzunza-CruzM Bricaire G Romero SZ Perez-Becker R Saavedra-Lira EPerez-Montfort R et al Leishmania mexicana mexicana genetic heteroge-neity of Mexican isolates revealed by restriction length polymorphismanalysis of kinetoplast DNA Exp Parasitol 200095277ndash84

[11] Mahboudi F Abolhassani M Tehrani SR Azimi M Asmar M Differen-tiation of old and new world Leishmania species at complex and specieslevels by PCR Scand J Infect Dis 200234756ndash8

[12] Noyes HA Reyburn H Bailey JW Smith D A nested-PCR-basedschizodeme method for identifying Leishmania kinetoplast minicircleclasses directly from clinical samples and its application to the study of theepidemiology of Leishmania tropica in Pakistan J Clin Microbiol1998362877ndash81

[13] Brewster M Barker DC Analysis of minicircle classes in Leishmania(Viannia) species Trans R Soc Trop Med Hyg 20029655ndash63

[14] Abranches P Pires CA Conceiccedilatildeo-Silva FM Silva-Pereira MCD Santos-Gomes GM Kala-azar em Portugal VIInqueacuterito epidemioloacutegico realizadona regiatildeo metropolitana de Lisboa interpretaccedilatildeo da estrutura e dinacircmica dofoco endeacutemico J Ciecircnc Meacuted Lisboa 1987151364ndash79

[15] Abranches P Sampaio-Silva ML Santos-Gomes G Avelino I Pires CAConceiccedilatildeo-Silva FM et al Kala-azar em Portugal VII Epidemiologicalsurvey in Alijoacute (Endemic region of Alto Douro) Res Ver Parasitol199252121ndash4

[16] Campino L Capela MJR Mauriacutecio IL Ozensoy S Abranches P O Kala-Azar em Portugal IX A regiatildeo do Algarve Inqueacuterito epidemioloacutegicosobreo reservatoacuterio canino no concelho de Louleacute Rev Port Doenccedilas Infecc199518189ndash94

[17] Semiao-Santos SJ el Harith A Ferreira E Pires CA Sousa C Gusmao REacutevora district as a new focus for canine leishmaniasis in Portugal ParasitolRes 199581235ndash9

[18] Campino L Pratlong F Abranches P Rioux J-A Santos-Gomes G Alves-Pires C et al Leishmaniasis in Portugal enzymatic polymorphism ofLeishmania infantum based on the identification of 213 strains TropMed Int Health in press

[19] Cortes S Rolatildeo N Ramada J Campino L PCR as a rapid and sensitivetool in the diagnosis of human and canine leishmaniasis using Leishmaniadonovani sl-specific kinetoplastid primers Trans R Soc Trop Med Hyg20049812ndash7

[20] Smyth AJ Ghosh A Hassan MQ Basu D De Bruijn MH Adhya S et alRapid and sensitive detection of Leishmania kinetoplast DNA from spleenand blood samples of kala-azar patients Parasitol 1992105183ndash92

[21] Martin-Sanchez J Gramiccia M Di Muccio T Ludovisi A Morillas-Marquez F Isoenzymatic polymorphism of Leishmania infantum insouthern Spain Trans R Soc Trop Med Hyg 200498228ndash32

[22] Marfurt J Niederwieser I Makia ND Beck HP Felger I Diagnosticgenotyping of Old and New World Leishmania species by PCR-RFLPDiagn Microbiol Infect Dis 200346115ndash24

[23] Guizani IG Van Eys JJM Riadh BI Koussay D Use of recombinant DNAprobes for species identification of old world Leishmania isolates Am JTrop Med Hyg 199450632ndash40

283S Cortes et al Parasitology International 55 (2006) 277ndash283

[24] Hide M Bantildeuls AL TibayrencM Genetic heterogeneity and Phylogeneticstatus of Leishmania (Leishmania) infantum zymodeme MON-1 epide-miological implications Parasitol 2001123425ndash32

[25] Pratlong F Dereure J Bucheton B El-Safi S Dessein A Lanotte G et alSudan the possible original focus of visceral leishmaniasis Parasitol20011222599ndash605

[26] Mauricio IL Gaunt MW Stothard JR Miles MA Genetic typing andphylogeny of the Leishmania donovani complex by restriction analysis ofPCR amplified gp63 intergenic regions Parasitol 2001122393ndash403

[27] Zemanova E Jirku M Mauricio IL Miles MA Lukes J Geneticpolymorphism within the Leishmania donovani complex correlation withgeographic origin Am J Trop Med Hyg 20047613ndash7

[28] Mauricio IL Stothard JR Miles MA The strange case of Leishmaniachagasi Parasitol 200016188ndash9

[29] Angelici MC Gramiccia M Gradoni L Study on genetic polymorphism ofLeishmania infantum through the analysis of restriction enzyme digestionpatterns of kinetoplast DNA Parasitol 198999301ndash9

[30] Mauricio IL Yeo M Baghaei M Doto D Pratlong F Zemanova E et alTowards multilocus sequence typing of the Leishmania donovani complexResolving genotypes and haplotypes for five polymorphic metabolicenzymes (ASAT GPI NH1 NH2 PGD) Int J Parasitol 200636757ndash69

[31] Lee ST Tarn C Chang KP Characterization of the switch of kinetoplastidDNA minicircles dominance during development and reversion of drugresistance in Leishmania Mol Biochem Parasitol 199358187ndash203

[32] Morales MA Chicharro C Ares M Cantildeavate C Barker DC Alvar JMolecular tracking of infections by Leishmania infantum Trans R SocTrop Med Hyg 200195104ndash7

Fig 3 Neighbor-joining tree using genetic distances derived from the kDNAPCR-RFLP data (genotypes) of all strains In black is the outgroup Close toeach genotype is the number of human (H) canine (C) and sand fly (S) isolatesGenotype G served as outgroup and included all African strains and aPortuguese MON-24 isolate ⋆ P ariasi P perniciosus

281S Cortes et al Parasitology International 55 (2006) 277ndash283

the relationships between the 15 genotypes Unfortunately boot-strapping was not appropriate and there is no measure of supportfor the groups found However the maximum likelihood tree (Lnlikelihood minus26463) was very similar with the exception of thecluster with genotypes D F K L M and N which had a differenttopology and also included genotype JWe have selected genotypeG as the outgroup given that it is present almost exclusively in Ldonovani strains from East Africa A Portuguese MON-24 wasalso found to have genotype G Genotype H is the closest to theroot and was found in a Portuguese MON-29 strain followed bygenotype O found in a MON-78 Maltese dermotropic strain Twomain clusters were detected with genotype B (one of the mostcommon in Portugal but present in Spain and Brazil) as a possiblecommon ancestor The cluster with genotypes E A C and I form avery clear line of descent from genotype B and have here onlybeen identified in Portugal

4 Discussion

kDNA has been exploited for diagnosis [2021] identifica-tion and genotyping of Leishmania species [10122223] In thepresent study the genetic diversity of Portuguese L infantumisolates has been evaluated using PCR-RFLP of kDNA Onehundred and twenty Portuguese isolates from humans dogs andsand flies from different regions of Portugal were analysed

The high number (13) of restriction profiles observed withinthe Portuguese samples indicates genetic heterogeneity withinthis parasite population Recent studies using different molecularmarkers and PCR-RFLP methods have uncovered genetic diver-sity within differentLeishmania species Cupolillo et al [6] usingRFLP analysis have found genetic diversity in Leishmaniabraziliensis from several regions of Brazil and most genotypeswere associated with specific geographic areas However in ourresults no genotype was exclusive to one focus perhaps because

our country is a small geographic region with migration betweenfoci

Two main genotypes (A and B) could be associated with theimmune status of the patients genotype A correlated with HIV+immunocompromised patients and B with immunocompetentpatients It is important to note that most of the LR isolates werefrom HIV+ drug addicts living in ghetto-like closed commu-nities inside Lisboa This could suggest that the population ofstrains circulating in AIDS patients may have suffered a foundereffect with propagation of a few clones This hypothesis willhave to be supported with a broader study

In the context of our total sample set within zymodemeMON-1 high diversity was observed in the sequence of kDNAanalysed as 11 restriction profiles were found Genetic diversitywithin MON-1 was also observed by others [24] In this studygenotype A the most frequent genotype was exclusive to thePortuguese population

The restriction profile was the same in all East African isolates(profile G) although they have been considered to belong todifferent zymodemes and species (L infantum L archibaldi andL donovani) [25] This finding corroborates other studies thatclassify the African strains as L donovani [2627] The presentstudy also shows the presence of one of the most frequent Portu-guese profiles (B) in the Brazilian samples Although just twoisolates have been studied this result reinforces the evidence thatL infantum and the New World parasite Leishmania chagasi areindeed synonymous species [28]

In this study we found that strains considered dermotropichad closely related genotypes (H and O) or the same genotype(G) as the outgroup strains Viscerotropic strains were heremonophyletic This is a similar result to that of Angelici et al[29] who showed that dermotropic L infantum strains weregenetically divergent from viscerotropic strains in a study ofkDNA-RFLP of 29 strains The Portuguese MON-24 strain withgenotype G (as present in the African L donovani strains) wasisolated from an intravenous drug user with AIDS It is possiblethat this strain was imported and that the kDNA genotype wasacquired by genetic exchange Such mosaic genomes have beenreported in the L donovani complex [30]

We have identified a line of descent of Portuguese geno-types formed by genotypes E A C and I in order fromgenotype B in which genotype Awas the most frequent one inthe Portuguese population It is possible that these genotypeshave evolved in Portugal The second cluster is much lessdefined and includes strains from other countries It is possiblethat these genotypes have evolved outside of Portugal and werethen imported The phylogenetic position of genotype J was notconsistent between the two tree building methods used Thisgenotype is the most distant from any other in this study and it islikely that the lack of intermediate genotypes complicates thephylogenetic analysis

In this study we assessed kDNA PCR-RFLP profile stabilitywith 4 sets of original isolates and after prolonged culture invitro after passage in vivo and with sequential isolates frompatients with several episodes of leishmaniasis Three of thestudied sets proved to be stable In one set we found a differentrestriction profile between the original isolate and after long-

282 S Cortes et al Parasitology International 55 (2006) 277ndash283

term in vitro culture indicating that the strain may have sufferedmutations in the kDNA sequences or changes in minicircle classfrequencies The change could be a consequence of adaptationdue to the pressure of long-time in vitro culture or due to agenetic driftfounder effect by sub-culturing Some authors haveproved that the mitochondrial genome can change in terms ofminicircle class frequencies due to in vitro drug exposure [31]On the other hand the results obtained on DNA of strainsisolated from patients that have had several episodes of leish-maniasis and revealed the same restriction profiles (in 11 of 12patients) confirms considerable stability among the minicirclesfrom sequential isolates before and after treatment The differentprofile observed in one case could correspond to a re-infectionrather than a relapse as was also suggested byMorales et al [32]However we cannot discard the possibility of having infectionsby mixed populations (or parasites with different frequencies ofminicircles) and a less dominant genotype becoming dominantafter treatment

We have found in the 3 phlebotomine isolates studied 3different profiles The parasites had been isolated from the twovector species (P ariasi and P perniciosus) in the country Twoof these genotypes (A B) were also identified from the samefocus in canine and human hosts According to Cupolillo et al[6] the presence of the same genotypes in humans and dogs inthe same area suggests that transmission cycles could be definedusing these methodologies

Thiswork also showed that the kDNAPCR-RFLPmethod canbe applied to parasite cultures or directly on biological samples

The results presented in this paper suggest that Portugal asmall country of South-eastern Europe considered homogenousfor the Leishmania species and zymodeme responsible forleishmaniasis presents more genetic diversity than previouslythought Further studies on L infantum diversity should becarried out with other genetic markers and analysed in conjunc-tion with our findings to reach firmer conclusions on the epi-demiology of leishmaniases in Portugal

Acknowledgments

We thank R Brazil (Centro de Pesquisas Reneacute Rachou Brazil)and C Chicharro (Instituto de Salud Carlos IIIMadrid Spain) forproviding us some of the strainsWe aremost grateful to cliniciansand pathologists from local hospitals that provided us with theclinical samples We would like to thank the European Com-mission (Contract no QLK2-CT-2001-01810) who supportedthis work and to all partners of the project with special regard tothe coordinator Michael Miles

References

[1] Dedet JP Pratlong F Leishmaniasis In Cook GC Zumla A editorsMansons Tropical Diseases London Saunders 2003 p 1339ndash64

[2] Alvar J Leishmaniasis and AIDS co-infection the Spanish exampleParasitol Today 199410160ndash3

[3] Campino L Santos-Gomes G Pratlong F Dedet JP HIV-Leishmania co-infection in Portugal isolation of Leishmania infantum MON-24 Trans RSoc Trop Med Hyg 199488394

[4] Tintaya KW Ying X Dedet JP Rijal S De Bolle X Dujardin JC Antigengenes for molecular epidemiology of leishmaniasis polymorphism of

cysteine proteinase B and surface metalloprotease glycoprotein 63 in theLeishmania donovani complex J Infect Dis 20041891035ndash43

[5] El Tai N El Fari M Mauricio IL Miles MA Oskam L El Safi SH et alLeishmania donovani intraspecific polymorphisms of Sudanese isolatesrevealed by PCR-based analyses and DNA sequencing Exp Parasitol20019735ndash44

[6] Cupolillo E Brahim LR Toaldo CB Oliveira-Neto MP Brito MEFalqueto A et al Genetic polymorphism and molecular epidemiology ofLeishmania (Viannia) braziliensis from different hosts and geographicareas in Brazil J Clin Microbiol 2003413126ndash32

[7] Mauricio IL Stothard JR Miles MA Leishmania donovani complexgenotyping with the ribosomal internal transcribed spacer and the mini-exon Parasitol 2004128263ndash7

[8] van Eys GJ Schoone GJ Kroon N Ebeling SB Sequence analysis of smallsubunit ribosomal RNA genes and its use for detection and identification ofLeishmania parasites Mol Biochem Parasitol 199251133ndash42

[9] Bulle B Millon L Bart JM Gallego M Gambarelli F Portus M et alPractical approach for typing strains ofLeishmania infantumbymicrosatelliteanalysis J Clin Microbiol 2002403391ndash7

[10] Berzunza-CruzM Bricaire G Romero SZ Perez-Becker R Saavedra-Lira EPerez-Montfort R et al Leishmania mexicana mexicana genetic heteroge-neity of Mexican isolates revealed by restriction length polymorphismanalysis of kinetoplast DNA Exp Parasitol 200095277ndash84

[11] Mahboudi F Abolhassani M Tehrani SR Azimi M Asmar M Differen-tiation of old and new world Leishmania species at complex and specieslevels by PCR Scand J Infect Dis 200234756ndash8

[12] Noyes HA Reyburn H Bailey JW Smith D A nested-PCR-basedschizodeme method for identifying Leishmania kinetoplast minicircleclasses directly from clinical samples and its application to the study of theepidemiology of Leishmania tropica in Pakistan J Clin Microbiol1998362877ndash81

[13] Brewster M Barker DC Analysis of minicircle classes in Leishmania(Viannia) species Trans R Soc Trop Med Hyg 20029655ndash63

[14] Abranches P Pires CA Conceiccedilatildeo-Silva FM Silva-Pereira MCD Santos-Gomes GM Kala-azar em Portugal VIInqueacuterito epidemioloacutegico realizadona regiatildeo metropolitana de Lisboa interpretaccedilatildeo da estrutura e dinacircmica dofoco endeacutemico J Ciecircnc Meacuted Lisboa 1987151364ndash79

[15] Abranches P Sampaio-Silva ML Santos-Gomes G Avelino I Pires CAConceiccedilatildeo-Silva FM et al Kala-azar em Portugal VII Epidemiologicalsurvey in Alijoacute (Endemic region of Alto Douro) Res Ver Parasitol199252121ndash4

[16] Campino L Capela MJR Mauriacutecio IL Ozensoy S Abranches P O Kala-Azar em Portugal IX A regiatildeo do Algarve Inqueacuterito epidemioloacutegicosobreo reservatoacuterio canino no concelho de Louleacute Rev Port Doenccedilas Infecc199518189ndash94

[17] Semiao-Santos SJ el Harith A Ferreira E Pires CA Sousa C Gusmao REacutevora district as a new focus for canine leishmaniasis in Portugal ParasitolRes 199581235ndash9

[18] Campino L Pratlong F Abranches P Rioux J-A Santos-Gomes G Alves-Pires C et al Leishmaniasis in Portugal enzymatic polymorphism ofLeishmania infantum based on the identification of 213 strains TropMed Int Health in press

[19] Cortes S Rolatildeo N Ramada J Campino L PCR as a rapid and sensitivetool in the diagnosis of human and canine leishmaniasis using Leishmaniadonovani sl-specific kinetoplastid primers Trans R Soc Trop Med Hyg20049812ndash7

[20] Smyth AJ Ghosh A Hassan MQ Basu D De Bruijn MH Adhya S et alRapid and sensitive detection of Leishmania kinetoplast DNA from spleenand blood samples of kala-azar patients Parasitol 1992105183ndash92

[21] Martin-Sanchez J Gramiccia M Di Muccio T Ludovisi A Morillas-Marquez F Isoenzymatic polymorphism of Leishmania infantum insouthern Spain Trans R Soc Trop Med Hyg 200498228ndash32

[22] Marfurt J Niederwieser I Makia ND Beck HP Felger I Diagnosticgenotyping of Old and New World Leishmania species by PCR-RFLPDiagn Microbiol Infect Dis 200346115ndash24

[23] Guizani IG Van Eys JJM Riadh BI Koussay D Use of recombinant DNAprobes for species identification of old world Leishmania isolates Am JTrop Med Hyg 199450632ndash40

283S Cortes et al Parasitology International 55 (2006) 277ndash283

[24] Hide M Bantildeuls AL TibayrencM Genetic heterogeneity and Phylogeneticstatus of Leishmania (Leishmania) infantum zymodeme MON-1 epide-miological implications Parasitol 2001123425ndash32

[25] Pratlong F Dereure J Bucheton B El-Safi S Dessein A Lanotte G et alSudan the possible original focus of visceral leishmaniasis Parasitol20011222599ndash605

[26] Mauricio IL Gaunt MW Stothard JR Miles MA Genetic typing andphylogeny of the Leishmania donovani complex by restriction analysis ofPCR amplified gp63 intergenic regions Parasitol 2001122393ndash403

[27] Zemanova E Jirku M Mauricio IL Miles MA Lukes J Geneticpolymorphism within the Leishmania donovani complex correlation withgeographic origin Am J Trop Med Hyg 20047613ndash7

[28] Mauricio IL Stothard JR Miles MA The strange case of Leishmaniachagasi Parasitol 200016188ndash9

[29] Angelici MC Gramiccia M Gradoni L Study on genetic polymorphism ofLeishmania infantum through the analysis of restriction enzyme digestionpatterns of kinetoplast DNA Parasitol 198999301ndash9

[30] Mauricio IL Yeo M Baghaei M Doto D Pratlong F Zemanova E et alTowards multilocus sequence typing of the Leishmania donovani complexResolving genotypes and haplotypes for five polymorphic metabolicenzymes (ASAT GPI NH1 NH2 PGD) Int J Parasitol 200636757ndash69

[31] Lee ST Tarn C Chang KP Characterization of the switch of kinetoplastidDNA minicircles dominance during development and reversion of drugresistance in Leishmania Mol Biochem Parasitol 199358187ndash203

[32] Morales MA Chicharro C Ares M Cantildeavate C Barker DC Alvar JMolecular tracking of infections by Leishmania infantum Trans R SocTrop Med Hyg 200195104ndash7

282 S Cortes et al Parasitology International 55 (2006) 277ndash283

term in vitro culture indicating that the strain may have sufferedmutations in the kDNA sequences or changes in minicircle classfrequencies The change could be a consequence of adaptationdue to the pressure of long-time in vitro culture or due to agenetic driftfounder effect by sub-culturing Some authors haveproved that the mitochondrial genome can change in terms ofminicircle class frequencies due to in vitro drug exposure [31]On the other hand the results obtained on DNA of strainsisolated from patients that have had several episodes of leish-maniasis and revealed the same restriction profiles (in 11 of 12patients) confirms considerable stability among the minicirclesfrom sequential isolates before and after treatment The differentprofile observed in one case could correspond to a re-infectionrather than a relapse as was also suggested byMorales et al [32]However we cannot discard the possibility of having infectionsby mixed populations (or parasites with different frequencies ofminicircles) and a less dominant genotype becoming dominantafter treatment

We have found in the 3 phlebotomine isolates studied 3different profiles The parasites had been isolated from the twovector species (P ariasi and P perniciosus) in the country Twoof these genotypes (A B) were also identified from the samefocus in canine and human hosts According to Cupolillo et al[6] the presence of the same genotypes in humans and dogs inthe same area suggests that transmission cycles could be definedusing these methodologies

Thiswork also showed that the kDNAPCR-RFLPmethod canbe applied to parasite cultures or directly on biological samples

The results presented in this paper suggest that Portugal asmall country of South-eastern Europe considered homogenousfor the Leishmania species and zymodeme responsible forleishmaniasis presents more genetic diversity than previouslythought Further studies on L infantum diversity should becarried out with other genetic markers and analysed in conjunc-tion with our findings to reach firmer conclusions on the epi-demiology of leishmaniases in Portugal

Acknowledgments

We thank R Brazil (Centro de Pesquisas Reneacute Rachou Brazil)and C Chicharro (Instituto de Salud Carlos IIIMadrid Spain) forproviding us some of the strainsWe aremost grateful to cliniciansand pathologists from local hospitals that provided us with theclinical samples We would like to thank the European Com-mission (Contract no QLK2-CT-2001-01810) who supportedthis work and to all partners of the project with special regard tothe coordinator Michael Miles

References

[1] Dedet JP Pratlong F Leishmaniasis In Cook GC Zumla A editorsMansons Tropical Diseases London Saunders 2003 p 1339ndash64

[2] Alvar J Leishmaniasis and AIDS co-infection the Spanish exampleParasitol Today 199410160ndash3

[3] Campino L Santos-Gomes G Pratlong F Dedet JP HIV-Leishmania co-infection in Portugal isolation of Leishmania infantum MON-24 Trans RSoc Trop Med Hyg 199488394

[4] Tintaya KW Ying X Dedet JP Rijal S De Bolle X Dujardin JC Antigengenes for molecular epidemiology of leishmaniasis polymorphism of

cysteine proteinase B and surface metalloprotease glycoprotein 63 in theLeishmania donovani complex J Infect Dis 20041891035ndash43

[5] El Tai N El Fari M Mauricio IL Miles MA Oskam L El Safi SH et alLeishmania donovani intraspecific polymorphisms of Sudanese isolatesrevealed by PCR-based analyses and DNA sequencing Exp Parasitol20019735ndash44

[6] Cupolillo E Brahim LR Toaldo CB Oliveira-Neto MP Brito MEFalqueto A et al Genetic polymorphism and molecular epidemiology ofLeishmania (Viannia) braziliensis from different hosts and geographicareas in Brazil J Clin Microbiol 2003413126ndash32

[7] Mauricio IL Stothard JR Miles MA Leishmania donovani complexgenotyping with the ribosomal internal transcribed spacer and the mini-exon Parasitol 2004128263ndash7

[8] van Eys GJ Schoone GJ Kroon N Ebeling SB Sequence analysis of smallsubunit ribosomal RNA genes and its use for detection and identification ofLeishmania parasites Mol Biochem Parasitol 199251133ndash42

[9] Bulle B Millon L Bart JM Gallego M Gambarelli F Portus M et alPractical approach for typing strains ofLeishmania infantumbymicrosatelliteanalysis J Clin Microbiol 2002403391ndash7

[10] Berzunza-CruzM Bricaire G Romero SZ Perez-Becker R Saavedra-Lira EPerez-Montfort R et al Leishmania mexicana mexicana genetic heteroge-neity of Mexican isolates revealed by restriction length polymorphismanalysis of kinetoplast DNA Exp Parasitol 200095277ndash84

[11] Mahboudi F Abolhassani M Tehrani SR Azimi M Asmar M Differen-tiation of old and new world Leishmania species at complex and specieslevels by PCR Scand J Infect Dis 200234756ndash8

[12] Noyes HA Reyburn H Bailey JW Smith D A nested-PCR-basedschizodeme method for identifying Leishmania kinetoplast minicircleclasses directly from clinical samples and its application to the study of theepidemiology of Leishmania tropica in Pakistan J Clin Microbiol1998362877ndash81

[13] Brewster M Barker DC Analysis of minicircle classes in Leishmania(Viannia) species Trans R Soc Trop Med Hyg 20029655ndash63

[14] Abranches P Pires CA Conceiccedilatildeo-Silva FM Silva-Pereira MCD Santos-Gomes GM Kala-azar em Portugal VIInqueacuterito epidemioloacutegico realizadona regiatildeo metropolitana de Lisboa interpretaccedilatildeo da estrutura e dinacircmica dofoco endeacutemico J Ciecircnc Meacuted Lisboa 1987151364ndash79

[15] Abranches P Sampaio-Silva ML Santos-Gomes G Avelino I Pires CAConceiccedilatildeo-Silva FM et al Kala-azar em Portugal VII Epidemiologicalsurvey in Alijoacute (Endemic region of Alto Douro) Res Ver Parasitol199252121ndash4

[16] Campino L Capela MJR Mauriacutecio IL Ozensoy S Abranches P O Kala-Azar em Portugal IX A regiatildeo do Algarve Inqueacuterito epidemioloacutegicosobreo reservatoacuterio canino no concelho de Louleacute Rev Port Doenccedilas Infecc199518189ndash94

[17] Semiao-Santos SJ el Harith A Ferreira E Pires CA Sousa C Gusmao REacutevora district as a new focus for canine leishmaniasis in Portugal ParasitolRes 199581235ndash9

[18] Campino L Pratlong F Abranches P Rioux J-A Santos-Gomes G Alves-Pires C et al Leishmaniasis in Portugal enzymatic polymorphism ofLeishmania infantum based on the identification of 213 strains TropMed Int Health in press

[19] Cortes S Rolatildeo N Ramada J Campino L PCR as a rapid and sensitivetool in the diagnosis of human and canine leishmaniasis using Leishmaniadonovani sl-specific kinetoplastid primers Trans R Soc Trop Med Hyg20049812ndash7

[20] Smyth AJ Ghosh A Hassan MQ Basu D De Bruijn MH Adhya S et alRapid and sensitive detection of Leishmania kinetoplast DNA from spleenand blood samples of kala-azar patients Parasitol 1992105183ndash92

[21] Martin-Sanchez J Gramiccia M Di Muccio T Ludovisi A Morillas-Marquez F Isoenzymatic polymorphism of Leishmania infantum insouthern Spain Trans R Soc Trop Med Hyg 200498228ndash32

[22] Marfurt J Niederwieser I Makia ND Beck HP Felger I Diagnosticgenotyping of Old and New World Leishmania species by PCR-RFLPDiagn Microbiol Infect Dis 200346115ndash24

[23] Guizani IG Van Eys JJM Riadh BI Koussay D Use of recombinant DNAprobes for species identification of old world Leishmania isolates Am JTrop Med Hyg 199450632ndash40

283S Cortes et al Parasitology International 55 (2006) 277ndash283

[24] Hide M Bantildeuls AL TibayrencM Genetic heterogeneity and Phylogeneticstatus of Leishmania (Leishmania) infantum zymodeme MON-1 epide-miological implications Parasitol 2001123425ndash32

[25] Pratlong F Dereure J Bucheton B El-Safi S Dessein A Lanotte G et alSudan the possible original focus of visceral leishmaniasis Parasitol20011222599ndash605

[26] Mauricio IL Gaunt MW Stothard JR Miles MA Genetic typing andphylogeny of the Leishmania donovani complex by restriction analysis ofPCR amplified gp63 intergenic regions Parasitol 2001122393ndash403

[27] Zemanova E Jirku M Mauricio IL Miles MA Lukes J Geneticpolymorphism within the Leishmania donovani complex correlation withgeographic origin Am J Trop Med Hyg 20047613ndash7

[28] Mauricio IL Stothard JR Miles MA The strange case of Leishmaniachagasi Parasitol 200016188ndash9

[29] Angelici MC Gramiccia M Gradoni L Study on genetic polymorphism ofLeishmania infantum through the analysis of restriction enzyme digestionpatterns of kinetoplast DNA Parasitol 198999301ndash9

[30] Mauricio IL Yeo M Baghaei M Doto D Pratlong F Zemanova E et alTowards multilocus sequence typing of the Leishmania donovani complexResolving genotypes and haplotypes for five polymorphic metabolicenzymes (ASAT GPI NH1 NH2 PGD) Int J Parasitol 200636757ndash69

[31] Lee ST Tarn C Chang KP Characterization of the switch of kinetoplastidDNA minicircles dominance during development and reversion of drugresistance in Leishmania Mol Biochem Parasitol 199358187ndash203

[32] Morales MA Chicharro C Ares M Cantildeavate C Barker DC Alvar JMolecular tracking of infections by Leishmania infantum Trans R SocTrop Med Hyg 200195104ndash7

283S Cortes et al Parasitology International 55 (2006) 277ndash283

[24] Hide M Bantildeuls AL TibayrencM Genetic heterogeneity and Phylogeneticstatus of Leishmania (Leishmania) infantum zymodeme MON-1 epide-miological implications Parasitol 2001123425ndash32

[25] Pratlong F Dereure J Bucheton B El-Safi S Dessein A Lanotte G et alSudan the possible original focus of visceral leishmaniasis Parasitol20011222599ndash605

[26] Mauricio IL Gaunt MW Stothard JR Miles MA Genetic typing andphylogeny of the Leishmania donovani complex by restriction analysis ofPCR amplified gp63 intergenic regions Parasitol 2001122393ndash403

[27] Zemanova E Jirku M Mauricio IL Miles MA Lukes J Geneticpolymorphism within the Leishmania donovani complex correlation withgeographic origin Am J Trop Med Hyg 20047613ndash7

[28] Mauricio IL Stothard JR Miles MA The strange case of Leishmaniachagasi Parasitol 200016188ndash9

[29] Angelici MC Gramiccia M Gradoni L Study on genetic polymorphism ofLeishmania infantum through the analysis of restriction enzyme digestionpatterns of kinetoplast DNA Parasitol 198999301ndash9

[30] Mauricio IL Yeo M Baghaei M Doto D Pratlong F Zemanova E et alTowards multilocus sequence typing of the Leishmania donovani complexResolving genotypes and haplotypes for five polymorphic metabolicenzymes (ASAT GPI NH1 NH2 PGD) Int J Parasitol 200636757ndash69

[31] Lee ST Tarn C Chang KP Characterization of the switch of kinetoplastidDNA minicircles dominance during development and reversion of drugresistance in Leishmania Mol Biochem Parasitol 199358187ndash203

[32] Morales MA Chicharro C Ares M Cantildeavate C Barker DC Alvar JMolecular tracking of infections by Leishmania infantum Trans R SocTrop Med Hyg 200195104ndash7