Human and canine leishmaniasis in asymptomaticand symptomatic population in NorthwesternGreece

C. Papadopouloua,*, A. Kostoulab, D. Dimitrioua, A. Panagioua,C. Bobojiannib, G. Antoniadesa

aZoonoses and Parasitology Unit, Department of Microbiology, Medical School, Dourouti UniversityCampus, University of Ioannina, 45110 Ioannina, GreecebClinical Microbiology Laboratory, University Hospital of Ioannina, Ioannina, Greece

Accepted 8 May 2004

Available online 20 July 2004

KEYWORDSLeishmaniasis

seroprevalence; Canine;

Human; Greece

Summary The occurrence of human and canine Mediterranean visceral leishmaniasis(MVL) in Northwestern Greece was investigated during an 8-year survey (1994–2001). Atotal of 1200 blood sera samples, collected from asymptomatic human population, and1200 blood sera samples from asymptomatic dogs were screened for Leishmaniainfantum antibodies using the indirect immunofluorescence test (IFA). Also during thesurvey 111 human subjects and 350 canines were referred as clinically suspect casesfor MVL.

Significant differences ðp ¼ 0:001Þ were found between the prevalence of MVL insymptomatic and asymptomatic human populations (12.6 and 0.5%, respectively), buta more modest difference was observed between symptomatic and asymptomaticcanines (45.4 and 24.4%, respectively). From the results, it appears that Greece has ahigh background of canine leishmaniasis, which is likely to be a risk factor for theemergence of human MVL.Q 2004 The British Infection Society. Published by Elsevier Ltd. All rights reserved.

Introduction

Visceral leishmaniasis (VL) has a wide distribution inthe tropical and subtropical regions. In addition, itis seen in the temperate zones of Asia and inendemic foci in the Mediterranean region. Greece isincluded among the European Mediterraneancountries, where endemic foci of VL occur since18th century. The Mediterranean VL (MVL) is a

zoonotic disease caused by Leishmania infantumidentified by isoenzyme electrophoresis fromhuman and canine isolates.1 Although, a clearinterdependence has not been observed betweenthe prevalence of human and canine leishmaniasis,dogs are believed to play an important role inhuman MVL transmission, with prevalences ofcanine infection ranging from 1.1% up to 48.4% indifferent Mediterranean countries.2 – 5 Clinicalmanifestations are observed only in a low pro-portion of infected dogs and serological diagnosisis considered essential for detecting the preva-lence of infection. In humans typical clinical

0163-4453/$30.00 Q 2004 The British Infection Society. Published by Elsevier Ltd. All rights reserved.doi:10.1016/j.jinf.2004.05.004

Journal of Infection (2005) 50, 53–60

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*Corresponding author. Tel.: þ30-26510 97592; fax: þ30-26510 93563.

E-mail address: cpapadop@cc.uoi.gr

manifestations are usually observed in youngerpatients entering the paediatric clinics with severesymptoms.

Although leishmaniasis in the Mediterraneanbasin is still mainly a disease of children, adultcases have been increasingly reported during thelast 20 years and HIV-MVL coinfection is beingreported more and more frequently in the Medi-terranean countries, especially in Spain, France andItaly.6 –12 In a comparative study of MVL among HIV-infected and non-HIV infected patients held duringa 24 years period in Spain, from 120 MVL casesdiagnosed, 80 (66%) were associated with HIVinfection, and the mean age of the infectedpatients was 33.2 vs. 23.2 yr of the non-HIVpatients.11

In Greece MVL was first reported in 1879 in theisland of Spetses12,13 and ever since there havebeen reports from various islands like Hydra,14,15

Crete and Zakynthos16 and from other coastal andinland regions like the greater Athens area in Atticaand the regions of Macedonia (Northern Greece)and Epirus (North western Greece).1,4,17 – 22

Although, the human MVL cases reported eachyear are few, the number of canine leishmaniasiscases reported is much larger and during the lastdecade the numbers of infected dogs are worryinglyincreasing.

Leishmaniasis is no longer confined to the poordeveloped countries and prevalence is increasingacross EU countries and USA. The current controlmeasures, including the effective vaccines and safedrugs are either no longer available or inadequate,while the number of risk factors (immune systemdisorders, HIV-infection, climate changes, popu-lation mass transportation for tourism, immigrationetc.) is increasingly making leishmaniasis a growingpublic health concern.23

In the present paper, we report data collected inthe region of Epirus in Northwestern Greece duringan 8-year survey launched by the Zoonoses andParasitology Units of the Department of Micro-biology, University of Ioannina, funded by the GreekMinistry of Research and Technology and theUniversity of Ioannina. Also the existing data ofhuman and canine leishmaniasis prevalence inGreece are compared to those available fromother Mediterranean countries.

Materials and methods

Study area

The four prefectures belonging to the Epirus region

and the near-by island of Leukada in the Ionian Seawere the areas surveyed during 1994–2001. Theprefectures surveyed were Ioannina, Arta, The-sprotia and Preveza, all coastal with the exceptionof Ioannina, a city in the mainland of Epirus, whichis built on the shores of a big Lake (Pamvotis Lake).The island of Leukada is next to Preveza and it isseparated from the mainland by a very narrowwater passage (width 20 m). The study area ispresented in Fig. 1.

Blood sampling

HumansA total number of 1200 blood sera samples fromasymptomatic, non-HIV positive, human population(ages 0–65 yrs) were collected. Three groups of 400samples each, equally distributed per age group,were collected from blood donors (ages 19–67 yrs),voluntarily participating healthy high school stu-dents (ages 13–18 yrs) and the Paediatric Clinic ofthe University Hospital of Ioannina (ages 0–12 yrs)from cases diagnosed as definite non-leishmaniasisand non-HIV cases. The patients visiting theUniversity Hospital of Ioannina came from North-western Greece (Epirus and Ionian islands). Thesamples collected from the Paediatric Clinic, theDonor Blood Bank and the high school students wereselected on the basis of their home address andnationality (only Greek), in order to have arepresentative sampling from the surveyed area.Also 111 serum samples were collected frompatients entering the University Hospital as clini-cally suspect leishmaniasis cases. According topatients records they came from different parts ofthe surveyed area and included members of thenative population and immigrants from southAlbania. The symptoms of the patients recordedas possible leishmaniasis cases included lymphade-nopathy, anorexia, weight loss, hepato-splenome-galy, pyrexia and abnormal blood counts (anaemia,leukopenia, thrombocytopenia). All suspectpatients were HIV negative, but most of them,especially the immigrant cases, were malnour-ished. All serum samples were stored in eppendorftubes at 220 8C until further processing.

DogsA total number of 1550 blood samples werecollected from domestic dogs by brachial veinpuncture. The sampled dogs were randomlyselected on the basis of permanently living in thesurveyed area. All dogs sampled served either forguarding or hunting and were kept outside thehouses of the owners. One thousand and twohundred sera samples were collected from

C. Papadopoulou et al.54

asymptomatic dogs. Collection of samples wasperformed in collaboration with the veterinaryauthorities throughout the surveyed area and thecollected samples were forwarded to the StateVeterinary Laboratory in Ioannina. The collectedsamples were accompanied by a record reportingthe breed, age, sex, place of birth, use (guarding,hunting, companion) of each dog, medical recordindicating whether the animal was healthy, immu-nosuppressed or clinically suspected of having anydisease including leishmaniasis, plus informationabout the owner (address, profession). Onlysamples from dogs assigned as healthy or notclinically suspect for leishmaniasis were selectedand forwarded to the Parasitology Unit of theMicrobiology Department, Medical School, Univer-sity of Ioannina. Also, 350 sera samples werecollected from dogs, which were diagnosed asclinically suspect for leishmaniasis in differentSmall Animal Practices at the surveyed area. Themost frequent symptoms in the clinically suspectdogs were peripheral lymphadenopathy, ulcerativelesions on the nose, ears and face, ocular lesions(conjunctivitis, blepharitis, uveitis) onychogrypo-sis, anorexia, wasting, weakness, chronic renalfailure, chronic myositis and hepato-splenomegaly.Sera were separated from all samples, transported

to the laboratory and stored in eppendorf tubes at220 8C until further processing.

Serological test

All blood sera samples collected were screened forL. infantum antibodies using the indirect immuno-fluorescence test (IFA). A commercially availabletest kit (Leishmania SPOT IF, BioMerieux) was usedfor screening both animal and human samplecategories, using anti-dog (Fluoline-dog, BioMer-ieux) and anti-human (Fluoline-H, BioMerieux) anti-sera, respectively. A series of two-fold serumdilutions ranging from 1:40 to 1:640 were, per-formed. Based on previous studies20 the surveyedarea is assumed as an endemic area for canineleishmaniasis, so a cut-off titre of 160 was chosen tobe considered positive for dogs and a cut-off titre of80 was considered to be positive for humans, apractice which has been followed by otherresearchers also.3,24 –26

Statistical analysis

The two-sided t-test was used for the statisticalevaluation of the results.

Figure 1 Map of the surveyed area. Numbers are showing the endemic foci of MVL. 1, Thesprotia; 2, Ioannina; 3,Preveza; 4, Arta; 5, Leukada island.

Human and canine leishmaniasis in Greece 55

Results

Human incidence

The screening of the healthy human populationrevealed six out of the 1200 humans tested to beseropositive, thus the prevalence in the asympto-matic seropositive humans was 0.5% (antibodytitres $1:80) (Table 1). From the six seropositivesubjects one (16.6%) was a child aged 10 years old(antibody titre ¼ 1:80) and five (83.3%) were adultsaged .50 years (antibody titre ¼ 1:160). No sig-nificant differences ðp ¼ 0:02Þ in relation to the ageand sex of the examined subjects were observed.Also 14 out of 111 cases suspected for leishmaniasis(12.6%) were serologically confirmed to be MVLinfected (antibody titres .1:160) (Table 1). Fromthe symptomatic seropositive human cases amasti-gotes were observed by microscopy in 4/14 cases(all adults) in stained smears after bone marrowbiopsy. Eight out of the 14 seropositive cases(57.1%) hospitalized at the University Hospital ofIoannina were immigrants from the neighboringvillages on the Albania side (South Albania) close tothe border with Greece and six out of the 14positive leishmania cases (42.8%) were natives fromthe Thesprotia Prefecture. No statistically signifi-cant difference was found in the prevalence ofinfection among immigrants and natives ðp ¼ 0:4Þ:However, all six native patients were habitants ofthe Filiates village, which is also near the border, onthe Greek side (Fig. 1). No significant difference inrelation to age was found ðp ¼ 0:02Þ although 10 outof the 14 (71.4%) leishmania cases hospitalized atthe University Hospital of Ioannina were childrenaged 1.5–8 years and only four out of the 14 cases(28.5%) were adults aged 25–40 years (Table 2). Inrelation to the sex of the infected subjects nosignificant differences ðp ¼ 0:5Þ were observedbetween males and females infected. All laboratoryconfirmed patients were treated with pentavalentantimonials (Glucantim, inject. solution 1.5 g) for15–20 days and in some cases (5%) the treatmenthad to be repeated after an interval of 2–4 weeks,because of sustaining high antibody titre. Allpatients administering Glucantim responded very

well to the treatment and recovered fully within $1month. No relapsing cases were recorded within thenext 3 years of follow-up of the patients.

Canine incidence

A total of 293 out of the 1200 asymptomatic dogstested were found to be seropositive (IFA antibodytitres $1:160), thus the prevalence of the seropo-sitive asymptomatic dogs was found to be 24.4%(Table 1). Ages of the seropositive dogs varied from6 months to 16 years and significant differencesðp , 0:001Þ in prevalence were observed in relationto age, with dogs aged over 8 years (67.5%) foundseropositive more frequently. Also 159 out of the350 dogs, which were diagnosed as clinicallysuspect for leishmaniasis at different Small AnimalClinics of the surveyed area were serologicallyconfirmed by IFA (antibody titres $1:160) and theprevalence was found to be 45.4% (Table 1). Ages ofthe infected dogs varied from 8 months to 15 yearsand significant differences ðp , 0:001Þ in preva-lence were observed in relation to age, the youngeranimals aged ,1 years (63.5%) been more vulner-able to the infection, with a male to female ratio1:2. Furthermore, a significant difference ðp ,

0:001Þ was observed in relation with the breed,the majority (63.5%) of the seropositive sympto-matic dogs were non-indigenous breeds (101/159)mainly German Shepherd, Dalmatian, Cocker,Collie, Setter, Doberman and Husky. IndigenousGreek breeds seemed to be more resistant to theleishmania infection, with an indigenous toimported breed ratio of infection 1:8, and theywere the majority (63.4%) of the asymptomaticcarriers (186/293) (Table 3).

Table 1 Results of IFA tests in humans and dogs by antibody titre

Antibody titres Asymptomatic humans Asymptomatic dogs Symptomatic humans Symptomatic dogs

1:40 18/1200 650/1200 14/111 191/3501:80 6/1200 484/1200 14/111 176/3501:160 5/1200 293/1200 14/111 159/3501:320 0/1200 291/1200 14/111 155/3501:640 0/1200 125/1200 12/111 155/350

Table 2 Age and nationality of seropositive symptomatichuman leishmaniasis cases

Nationality Number of positive cases (%)

Greeks 6/14 42.8Albanians 8/14 57.1Children 1.5–8 yrs 10/14 71.4Adults 4/14 28.5

C. Papadopoulou et al.56

Geographical considerations

In the surveyed area certain foci of VL wereidentified. The highest prevalence of leishmaniainfection was detected both in the dog (82.8%) andthe human (42.8%) symptomatic population inThesprotia prefecture (Filiates village) near theborderline between Greece and Albania (Table 4).Next higher prevalence for canine leishmaniasis wasfound in the Ioannina prefecture, which wasfollowed by the Arta and Preveza prefectures andlastly the island of Leukada (Figs. 1 and 2).

Discussion

In the present survey conducted in NorthwesternGreece significant differences ðp ¼ 0:001Þ werefound between the prevalence of leishmaniasis insymptomatic and asymptomatic human populations(12.6 and 0.5%, respectively, a 25-fold difference),but a more modest difference was observedbetween symptomatic and asymptomatic dogs(45.4 and 24.4%, respectively, a two-fold differ-ence). Previous studies from Greece have reportedprevalences ranging from 0.5 to 9.2% in asympto-matic humans17,20 and 14.8 to 33.4% in symptomaticpatients.18,20 In asymptomatic dogs previousstudies indicated a seroprevalence ranging from10.8 to 24.0%,17,20,21,28 and from 40 to 48.4% insymptomatic animals.4,17,20 It is known that themethod chosen for detection of leishmania in

symptomatic dogs can affect results, with IFATand PCR giving estimates of 12.3 and 63% positivereadings.28

Results from this study conducted in Greececompare favorably to those observed in otherMediterranean countries. Two studies conductedin Turkey indicated canine seroprevalences of 5.3and 3.6%;5,29 childhood MVL is endemic in Turkey(mean age 3 years; 40 cases reported from 1981 to2001.30 In Cyprus canine seroprevalence ratesranged from 12.2 to 26.2%, but only 1.7% of dogsreacted positively when tested for L. infantumantibodies by enzyme linked immunosorbent assay(ELISA).31

Increasing canine leishmaniasis has beenrecorded in Italy (western Sicily and Aeolian islands)since the late 1980’s, but only a few cases of MVLhave been reported in children and in adults duringthat time frame.32 However, young children (meanage of 1.7 years) and severely malnourishedchildren are at risk for leishmaniasis in this country,as revealed by a retrospective analysis of reportedcases in Italy from 1980 to 2000.33 The Sicilianprovince of Catania is an active focus of humanMVL, with 10–15 cases diagnosed per year; how-ever, as many as 37–38 cases have been reported insome years.34 The childhood MVL incidence in Maltais 0.9 per 100 000, capita, as measured from 1994 to1998. In contrast, paediatric incidence is 2.5 per100 000 capita, and the median age at presentationin this study was 34 months.35 Canine seropreva-lence in Corsica was 18.6% in one study.36 In France,canine, leishmaniasis ranged from 0.23 to17.13%.37,38 A very high incidence of dog cases hasbeen noticed in Marseilles, but only a few peoplewere noted to develop the disease. Interestingly,65% of the tested human subjects in this regionwere sensitized against L. infantum.39 In SouthernSpain, 5.3% of asymptomatic dogs were positive,whereas 44.16% of asymptomatic humans werepositive to skin test for antibodies to L. infantum.3

In Tunisia, paediatric MVL prevails and 62.5% ofcases are associated with malnutrition, whichincluded an increase from 10 to 50 cases per yearfrom 1992 to 1994; suggested risk factors for the

Table 3 Prevalence of canine and human leishmaniasis reported from different regions of Greece

Greece Dogs Humans

Region investigated Asymptomatic (%) Symptomatic (%) Asymptomatic (%) Symptomatic (%)

Athens Greater area (Southern Greece) 22.421 48.44 33.427

Macedonia (Northern Greece) 10.817 40.017 9.217

Epirus (Northwestern Greece) 24.420 45.420 0.5 12.6Thessaly (Central Greece) 12.328

Table 4 Prevalence of canine leishmaniasis in asympto-matic dogs in different European countries

Country Asymptomatic canine VL (%)

Cyprus 12.2–26.231

France 0.23–18.636–38

Greece 10.8–48.44,17,20

Italy (Sicily) 60.034

Portugal 9.6–29.425

Russia 19.142

Spain 5.3–9.240

Turkey 3.6–5.35,29

Human and canine leishmaniasis in Greece 57

increase were increased rainfall and a change inagricultural practices.41 In Russia, 8.0% of asympto-matic humans and 19.1% of healthy dogs yieldedpositive results.42

Evidence drawn from the literature and specifi-cally from the studies mentioned above suggeststhat the relationship between canine and humanleishmaniasis is complicated. Thus, some evidencesuggests that dogs are a reservoir for at-riskpaediatric and immunosuppressed human popu-lations following transmission through sandflies.However, it also appears that high percentages ofboth canines and humans seroconvert withoutdeveloping symptoms, which may indicate a highlevel of exposure and population immunity in bothspecies. Only one study implicated dogs in thetransmission of leishmaniasis to humans by detect-ing identical strains in both species; however,further studies would be needed to determine atwhat rate immunological seroconversion isaccompanied by illness in humans. It is noted thatindigenous canine breeds may be more resistant todeveloping symptomatic leishmaniasis. In thisstudy, the ratio of indigenous to imported breedswas 1:8, but most asymptomatic carriers (63.4%)were indigenous breeds. In healthy appearing dogsfrom the United States that had been stationed inSicily, 60% of animals had elevated IgM antibodylevels suggesting that they had been exposed to L.infantum.34

In comparison to other Mediterranean countries,Greece appears to have a high background of canineleishmaniasis, second only to that of Sicily. It is thuslikely that high incident canine leishmaniasis is arisk factor for the emergence of MVL. Other riskfactors may include: (1) the warming climate in theSoutheastern Mediterranean countries that

enhances the propagation of the sandfly popu-lation,43 (2) changes in agricultural pesticidepractices that in the past suppressed sandflypopulations, (3) an increase in the stray dogpopulation in Greece, (4) an increase in importeddog populations, (5) a change in immigrant popu-lations beginning in 1990, and (6) ineffectivechemotherapeutic management of symptomaticdogs.44 Controlling stray dog populations and sand-fly levels is difficult to implement because of animalwelfare and environmental concerns. However, it isclear that the medical community should helpeducate those at most risk of developing MVL,which includes the malnourished, the very youngand immunosuppressed patients how to avoidexposure to sandflies and how to recognize thesymptoms of leishmaniasis.

Acknowledgements

This study was funded by the General Secretariat ofResearch and Technology of the Greek Ministry ofDevelopment and the University of Ioannina funds.The authors wish to thank all the veterinariansworking in the public services and the private sectorin the surveyed area of Northwestern Greece fortheir cooperation in the collection of dog blood serasamples. Special thanks are expressed to the formerdirector of the State Veterinary Laboratory atIoannina, Greece, Mr E. Manis and his colleaguesMrs C. Papachristodoulou and P. Tsimogianni. Also,we wish to express sincere thanks to ProfessorP. Lapatsanis, former Director of the PaediatricClinic and Dr E. Zervou, Director of the Blood Bankof the University Hospital of Ioannina for kindlyproviding the blood donor’s serum samples. We

Figure 2 Incidence of canine leishmaniasis by prefecture.

C. Papadopoulou et al.58

wish to thank Assistant Professor A. Bartzokas fromthe Laboratory of Meteorology, Department ofPhysics, University of Ioannina, for his helpfulcomments and useful information on climaticchanges in Greece during the survey period. Alsowe would like to thank all the doctors, nurses, civil,authorities, high school teachers, Parent associ-ations and the high school students for voluntarilyparticipating in this study and helping us to realizethis survey.

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