Travel Medicine and Infectious Disease (2011) 9, 37e46

ava i lab le at www.sc iencedi rec t .com

journa l homepage: www.e lsev ierhea l th .com/ journa ls / tmid

Manifestations of paediatric Leishmania infantuminfections in Malta

David Pace a,*, Thomas N. Williams b, Alicja Grochowska c, Alexandra Betts d,Simon Attard-Montalto e, Michael J. Boffa f, Cecil Vella e

a Paediatric Infectious Diseases Clinic, Mater Dei Hospital, Msida, MaltabDepartment of Paediatrics, Oxford and KEMRI Wellcome Trust Unit, Kilifi, KenyacDepartment of Haematology, Mater Dei Hospital, Msida, MaltadDepartment of Histopathology, Mater Dei Hospital, Msida, MaltaeDepartment of Paediatrics, Mater Dei Hospital, Msida, MaltafDepartment of Dermatology, Sir Paul Boffa Hospital, Floriana, Malta

Received 24 August 2010; received in revised form 22 November 2010; accepted 30 November 2010Available online 5 January 2011

KEYWORDSPaediatricleishmaniasis;Leishmania infantum;Phlebotomine sand fly;Malta

* Corresponding author. Paediatric InMalta. Tel.: þ356 2545 5558; fax: þ35

E-mail address: dpace@go.net.mt

1477-8939ª 2010 Elsevier Ltd.doi:10.1016/j.tmaid.2010.11.005

Open ac

Summary Leishmania infantum is endemic in the Maltese archipelago, a group of islands inthe Mediterranean which are visited frequently by tourists from Northern European countries.The burden of leishmaniasis is highest in children who may present with cutaneous or visceralmanifestations. We describe systematically the manifestations, diagnosis and management ofleishmaniasis in children <14 years of age, who had a histopathological diagnosis of leishman-iasis in Malta, from 2004 to 2008. Eleven children were diagnosed with leishmaniasis; 8 children(15e44 months of age) had visceral disease and three (aged 9e13 years) suffered cutaneousinfections. Prolonged high grade fever, pallor, hepatosplenomegaly, and pancytopenia werecommon presenting features of visceralisation. Diagnosis was based on the visualisation ofamastigotes from bone marrow aspirates. Pentavalent antimonials were associated with treat-ment failure in two children, whilst liposomal amphotericin B was curative in all. Children withcutaneous leishmaniasis had dry crusted ulcero-nodular lesions on exposed areas which re-sponded to intra-lesional instillation of sodium stibogluconate or to cryotherapy. Leishmaniasisshould be included in the differential diagnosis of fever and hepatosplenomegaly or chroniccutaneous lesions in children who travel to Malta.ª 2010 Elsevier Ltd. Open access under CC BY license.

fectious Diseases Clinic, Department of Paediatrics, Mater Dei Hospital, Tal-Qroqq, Msida, MSD 2090,6 2545 4154.(D. Pace).

cess under CC BY license.

38 D. Pace et al.

Introduction

The leishmaniases are a group of vector-borne protozoandiseases caused by pathogenic Leishmania species which, ifsymptomatic, result in protean clinical manifestations thatrange from localised cutaneous ulcers to disseminated lethalinfection. Such diversity is complicated by anthro-ponozoonotic modes of transmission which follow a sylvatic,peridomestic or anthroponotic cycle depending on themammalian reservoir.1 Being widespread in all continents(except Australia and Antarctica), but primarily concen-trated in South Asia, the Horn of Africa, Central and SouthAmerica and endemic in the Mediterranean basin, theseparasitoses are a major public health concern.2 Globalannual incidence is estimated at 1.5 million cases of cuta-neous disease and 500,000 cases of visceral leishmaniasis(VL), with prevalence rates reaching 12 million.2 Leishman-iasis is estimated to result in a loss of 1.97 million disabilityadjusted life years (DALYs) worldwide, classifying third fromall vector-borne infections.3 In Southern Europe annualincidence rates of VL, ranged from 0.11 to 8.32/100,000population from 1998 to 2007.4 In non-endemic countriesleishmaniasis is a disease of travellers and migrants.5

Leishmaniasis is endemic in the Maltese archipelago,a group of small islands (consisting of Malta, Gozo andComino) in Southern Europe with an area of 315 km2,a population of around 400,000 inhabitants and a pop-ulation density of 1285 residents per km2, the highest in theEuropean Union.6 Malta is visited by an average of 1.2million tourists per year, most of whom come from northernEuropean countries, namely UK and Germany.6 Cutaneousand visceral leishmaniases in Malta are caused by a singlespecies, Leishmania infantum, which is transmitted fromdogs to humans by one local species of sand fly, Phleboto-mus perniciosus.7 The few studies performed in Malta havenot identified any other Leishmania species in humans whoacquired cutaneous disease in Malta, or in sand flies, dogsor rats8,9 and because of limited resources species identi-fication from clinical specimens is not carried out as yet.

InMalta twozymodemes (strainswithdifferent isoenzymeprofiles) of L. infantum have been characterised: MON1 thatcauses VL and, as in other Mediterranean countries, is thecommoner form, and MON78 which is dermotropic,8 butwhich may cause VL in the immunocompromised.10 Notifi-cation of leishmaniasis has been compulsory in Malta since1946, however LCLhadnot been recognised prior 1981 andnocases were notified before 1983.11 By contrast, VL had beenrecognised since 1909,12 with the largest number of cases(207), being recorded in 1948.13 The quasi-eradication ofstray dogs, improved sanitary conditions and urbanisationhave resulted in a drastic decrease in the incidence of VLfollowing the SecondWorldWar, from67/100,000 populationin 1948 to 8.2/100,000population in 1955 anddown to ameanof 1.1/100,000 over the last 10 years.13 Rates of cutaneousdisease have remained low since 1983 (mean 1.87/100,000),14 however, being a non-life-threatening illness andoften not notified or unrecognised, the incidence of LCL isconfounded by underreporting. Children <3 years of agesuffer themajority of the visceral disease burden.15 The casefatality rate is extremely low with only two individuals, aged>65 years, dying from VL since 1991.16 Leishmaniasis has not

yet been eradicated due to persistent transmission fromcanine leishmaniosis,9 the only identified reservoir in Malta,with 31% (406/1310) of all indirect immunofluorescence (IF)tests carried out from 2005 to 2008 on dogs with suspectedleishmaniosis being positive.17 The prickly pear (Opuntiavulgaris) and rubble walls, which are widespread in Malta,create a perfect habitat for the breeding of sand flies.18

The female phlebotomous sand fly, a 2e3 mm arthropodbelonging to the genus Phlebotomus in the Old World isa noiseless flier which feeds from dusk throughout thenight.19 Outdoor activities are common in Malta during hotsummer nights (the peak tourist season), putting unawareindividuals at risk of being bitten by P. perniciosus, whoseprobing activity is increased when infected.20 Age relateddifferences in T-cell immunity might contribute to the highdisease burden in children <3 years old. Seasonal variationof leishmaniasis has not been observed in Malta due to itsvariable incubation period (6 weekse6 months but at timesas long as 10 years).15

We describe the manifestations, diagnosis and manage-ment of leishmaniasis in children, diagnosed in Malta overa 5 year period, from 2004 to 2008, in order to makephysicians in non-endemic countries aware of the persis-tence of leishmaniasis in this Mediterranean country.Leishmaniasis has a wide differential diagnosis and is animportant treatable parasitosis that should be considered inpaediatric travellers returning from endemic regions.

Methods

The hospital records of all children (<14 years) witha histopathological diagnosis of leishmaniasis from 2004 to2008, who were identified from the database of thePathology Department at Mater Dei Hospital which is themain acute general hospital in Malta, were reviewed. Eightchildren (aged 15e44 months) presented with visceralleishmaniasis, of which 7 were female. Six children wereMaltese (4 from Malta and 2 from Gozo), and 2 were Somali(Table 1). The two refugees from Somalia were a boy aged44 months, who had been living in Malta for the previous 11months, and an 18 month old girl who had been in Malta for1 year. Three Maltese children (aged 9e13 years) hada definite histological diagnosis of cutaneous leishmaniasis(Table 2). All were male and one lived in Gozo. A singleulcerated nodule was noted below the left lower eyelid inthe 13 year old boy (Case 10) and on the right arm in the 11year old boy (Case 11). The 9 year old Gozitan boy (Case 9)had multiple lesions on his face and right lower limb.

Results

Visceral leishmaniasis

Clinical featuresProlonged high grade fever was observed consistently in all 8children presenting with VL. Other symptoms includedanorexia, irritability, cough, rhinorrhoea, vomiting anddiarrhoea. All appeared pale and hepatosplenomegaly wasdetected in all, except inCase 5,whoonly had splenomegaly.None exhibited prominent lymphadenopathy. Most children

Table 1 Presentation and management of children with visceral leishmaniasis (2004e2008).

Case number Year Age/months

Sex Presentation Investigations Treatment Bloodproducts

Complications Hospitalstay/days

Case 1 Maltese 2004 15 F Febrile up to 40.5 �Cfor 10 daysPallorHepatosplenomegaly

PancytopeniaCRP: 143 mg/lBM: LDBs

Sodium stibogluconate þallopurinol � 10 days(switched to Ambisome[L-AmB] after 20 days)

PCPlateletsalbumin

� Declining platelet countsdown to 2 � 109/l, causingepistaxis, diminishing haemo-globin concentration despitetransfusion, non-resolvinghepatosplenomegaly, devel-opment of ascites and persis-tence of fever >38 �C after 20days of antimonials indicatedtreatment failure: switched toL-AmB for 10 days (20 mg/kg)

� L-AmB induced hypokalaemia

30

Case 2 Gozitan 2005 27 F Febrile up to 39.4 �Cfor 7 daysPallorHepatosplenomegalyPharyngitis

AnaemiaNeutropeniaCRP: 207 mg/laELISA IgG/IgM: 20.31BM: LDBs

Sodium stibogluconate þallopurinol � 10 days(switched to L-AmB after14 days)

PC � Generalised macular rash andrecurrence of fever after 10days of antimonials associatedwith pus oozing from cannulasite. Diagnosed with caMRSAbacteraemia and treated withteicoplanin for 10 days

� Non-resolving fever 5 daysthrough teicoplanin andenlarging spleen indicatedtreatment failure: antimonialsswitched to L-AmB for 10 days(30 mg/kg)

18

Case 3 Somalia(in Malta for11 months)

2006 44 M Febrile up to 40 �C for 7daysDiarrhoeaPallorHepatosplenomegaly

AnaemiaThrombocytopeniaCRP: 132 mg/lIF IgG þ ve, BM: LDBs

Sodium stibogluconate for21 daysAllopurinol for 10 days

PC � ANA pos 1:80 (fine specklednucleolar pattern): notdetected 3 months later

24

Case 4 Gozitan 2007 28 F 7 day h/o fever up to40�CClear rhinorrhoeaMiserablePallorHepatosplenomegaly

AnaemiaThrombocytopeniaCRP:187 mg/l,ESR:72 mm/hIF IgG: þveBM: LDBs

Sodium stibogluconate for28 daysAllopurinol for 10 days

Nil Nil 11

Case 5 Maltese 2007 19 F Febrile up to 39 �C for 4daysPallorSplenomegaly

Pancytopenia:CRP:100 mg/l,ESR: 56 mm/hELISA IgG/IgM: 20.3,BM: LDBs

Sodium stibogluconate for21 daysAllopurinol for 10 days

PC Nil 5

(continued on next page)

Leish

maniasis

inch

ildrenin

Malta

39

Table 1 (continued)

Case number YearAge/months Sex Presentation Investigations Treatment

Bloodproducts Complications

Hospitalstay/days

Case 6 Maltese 2007 16 F Recurrent URTIs duringthe previous 4 monthsPoor weight gain,Febrile up to 38.2 �C inhospital,IrritablePallorHepatosplenomegaly

PancytopeniaDirect Coombs test:þveBM: LDBs

L-AmB for 10 days(30 mg/kg)

PC Nil 4

Case 7 Maltese 2008 20 F Febrile up to 40.2 �Cfor 7 daysRigorsIrritablePallorHepatosplenomegaly

PancytopeniaCRP 29 mg/lIF IgG þ veELISA IgG/IgMeveBM: LDBsPCR: L. infantum þve

L-Amb for 10 days(20 mg/kg)

Nil Nil 11

Case 8 Somalia(in Malta for1 year)

2008 18 F Febrile up to 40.3 �Cfor 3 weeksRefusing to walkPharyngitis,Hepatosplenomegaly

PancytopeniaCRP 114 mg/lIF IgG þveELISA IgM/IgG: 20.7BM: LDBs

L-Amb for 10 days(20 mg/kg)

PC Nil 11

Abbreviations: M: male, F: female, URTI: Upper respiratory tract infection, CRP: C-reactive protein, BM: Bone marrow aspirate, LDBs: Leishman-Donovan bodies, ELISA: Enzyme-LinkedImmunosorbent Assay, IF: Indirect immunofluorescence assay for L. infantum IgG; ESR: Erythrocyte sedimentation Rate, PCR: polymerase chain reaction, PC: packed cells, ANA: Anti-Nuclear Antibodies.a Values for ELISA represent an antibody index with a cut off >11 taken as positive.

40D.Pace

etal.

Table

2Presentationandmanage

mentofch

ildrenwithcu

taneousleishmaniasis(200

4e20

08).

Case

number

Year

Age

/years

Sex

Presentation

Inve

stigations

Histology

Treatm

ent

Case

9Gozitan

2005

9M

2month

h/o

multiple

enlargingroundnodules

onlateralborderrigh

teye

,righ

ttemporalarea,

left

cheek,

left

pinna,righ

tthighandkn

ee.

Lesionscrustedin

centreandocc

asionallyooze

d.

Punch

biopsy

form

lesions

ontemple

andch

eek

Seve

ralgranulomata

andLD

Bsin

histiocytes

Cryotherapy�2

,ata

2month

interval,

usingliquid

nitroge

n

Case

10Maltese

2007

13M

2ye

arh/o

painless

crustedulceratednodule

1.4cm

indiameter,

below

theleft

lowereye

lid

Exc

isedunderloca

lanaesthesia

Granulomatous

inflammationwithLD

Bs

Nofurthertreatm

ent

Case

11Maltese

2008

11M

3month

h/o

painless

crustedulceratednodule,

1cm

indiameter,

onthelateralaspect

ofthe

righ

tarm

Slit

skin

smears

followed

bypunch

biopsy

Cytology

ofskin

smears:

lymphocy

tes,

macrophage

sandmulti-nuce

latedgiantce

lls

Biopsy:tuberculoid-typ

egranulomas,

scatteredLD

Bs

intra-lesionalsodium

stibogluco

nate

�4at5dayintervals

Abbreviations:

M:male,h/o

:history

of;

LDBs:

Leishman-Donova

nbodies.

Leishmaniasis in children in Malta 41

appeared well between episodes of fever and none had signsof septic shock or disseminated intravascular coagulation.

Laboratory findingsPancytopenia was a frequent but inconsistent finding withtotal white cell counts of 2.3e5.9 � 109/l, neutrophils aslow as 0.22 � 109/l (range 0.22e2.8 � 109/l), haemoglobinconcentration of 5.3e9.5 g/dl with microcytic, hypo-chromic red cells, and platelet counts of 34e157 � 109/l.High C-reactive protein (range: 29e207 mg/l), an ESR>40 mm/h, hypoalbuminaemia and negative blood cultureswere observed consistently. A bone marrow aspirate wasdiagnostic in all (Fig. 1) and excluded any underlying hae-matological malignancy. Serological testing using an IFassay for anti-L. infantum IgG (Leishmania-Spot IF, bio-Merieux, Marcy l’Etoile, France)21 and/or an indirectenzyme-linked immunosorbent assay (ELISA) for anti-L.infantum IgG þ IgM (Vircell, S.L., Granada, Spain)22 wereperformed. All children tested had a positive IF assay butELISA was falsely negative in Case 7 who, subsequently, wasconfirmed to be infected with L. infantum using a poly-merase chain reaction (PCR) assay detecting the speciesspecific genome regions SSUrRNA and ITS-1. The two Somalirefugees were presumed to have acquired the infection inMalta; however, species identification was not performed.Anti-nuclear antibodies were detected transiently in case3.

TreatmentSodium stibogluconate (Pentostam, GlaxoSmithKline, Mid-dlesex, UK) at a dose of 20 mg/kg for 21 days (preceded byan initial test dose of 25 mg) in combination with oralallopurinol (20 mg/kg daily for 10 days) was the treatmentof choice prior 2007. Liposomal amphotericin B (L-AmB,AmBisome, Gilead Sciences Int. Ltd, Cambridge, UK) ata total dose of 20e30 mg/kg administered over 10 days, waspreferred subsequently in view of concerns on treatmentfailure with antimonials. Most children received a bloodtransfusion for anaemia since the haemoglobin concentra-tion is known to drop in the first 2 weeks of treatment.15

The microcytosis, possibly resulting from associated irondeficiency, generally resolved after treatment and nonehad an underlying haemoglobinopathy. Case 1 neededmultiple platelet transfusions due to recurrent epistaxis asa result of thrombocytopenia (dropping to 2 � 109/l) asso-ciated with treatment failure. Because of the underlyingneutropenia, many children were started on antibiotics onadmission. The caMRSA bacteraemia in Case 2, resultingfrom an infected intravenous cannula, was associated withan appropriate neutrophilic response.

Response to treatmentSuccessful treatment resulted in defervescence (notedwithin 2e8 days) which coincided with a rise in the plateletcount, whilst unremitting fever or its recrudescence,a decreasing platelet count or non-resolving hep-atosplenomegaly indicated treatment failure (Case 1: a 15month old Maltese girl and Case 2: a 27 month old girl fromGozo: Table 1). Hospital stay for children showing animmediate response was short (4e11 days) and the fulltreatment course was subsequently completed as outpa-tients. Because of the language barrier and social

Figure 1 Bone marrow smears from Case 3 (Giemsa stain �1000). a) Intra-macrophageal Leishman-Donovan bodies; b) Rupturedmacrophage with release of several amastigotes, c) Extracellular amastigote with prominent nucleus and kinetoplast, d) Cyst-likestructure (arrow) containing amastigotes. Source: Haematology Department, Mater Dei Hospital, Malta.

Figure 2 Cutaneous leishmaniasis (Case 11): Raisederythematous nodule (diameter of 1 cm) with central dryulceration and surrounding inflammatory hypopigmentation onthe right lateral arm. Source: MJB, Dermatology Department,Sir Paul Boffa Hospital, Floriana, Malta.

42 D. Pace et al.

background, the two Somali children were kept in hospitalto ensure treatment compliance. Treatment failure wasassociated with a prolonged hospital stay (18e30 days).Despite their documented toxicities, no complications wereassociated with antimonials and the hypokalaemiaobserved in Case 1 was induced by L-AmB. All children werefollowed up for 6e12 months and none developed anyclinical signs of relapse.

Localised cutaneous leishmaniasis (LCL)

Clinical presentationThe three boys presenting with cutaneous disease hadcrusted ulcero-nodules (<1.5 cm in diameter) on exposedareas namely on the face, arm (Fig. 2) and legs, with Case 9having multiple lesions (Table 2). Diagnosis was delayed inCases 9 and 10 for 3 months and 2 years, respectively sincethe disease was not recognised, whilst Case 11 waspromptly diagnosed at presentation. Case 9 received localantimicrobial treatment for suspected fungal lesions andwas also prescribed oral flucloxacillin for possible impetigo.Similar treatment was administered to Case 10 on whomincision and curettage of the ‘cyst-like’ lesion was alsoinappropriately attempted with no success.

Laboratory findingsHistological examinationof tissue specimenswas diagnostic inall. Diagnosis of LCL in Case 10 (Fig. 3) was only clinched afterhis skin lesion was ultimately excised, 2 years after onset, for

psychological distress. Slit skin smears performed on case 11did not reveal the Leishmania amastigotes, however, similarto Case 9, a punch biopsy of the lesion was diagnostic.

TreatmentThe management of all three cases was different: Case 9received cryotherapy with liquid nitrogen in view of the

Figure 3 Histology of cutaneous leishmaniasis in Case 10 (H&E stain): a) Diffuse chronic inflammatory cell infiltrate in dermiswith multiple non-caseating granulomata (�40); b) Tuberculoid-type granulomata with central histiocytes and peripheralinflammatory cells (�100); c) Leishman-Donovan bodies (arrows) within cytoplasm of epithelioid histiocytes (�600); d) Langhanstype giant cells (arrows) within a granuloma (�600). Source: Pathology Department, Mater Dei Hospital, Malta.

Leishmaniasis in children in Malta 43

multiplicity of his lesions, the surgical procedure carriedout on Case 10 was curative and Case 11 received repeatedintra-lesional injections of sodium stibogluconate. Alltreatment modalities resulted in clinical cure and althoughCase 10 had a residual surgical scar he had no recurrence.

Discussion

The clinical manifestations of the children described in ourstudy are typical of the presentation of childhood VL indeveloped countries in the Mediterranean littoral.23e26 Bycontrast, VL in children in Albania, a less industrialisedMediterranean countrywith amuch higher incidence rates ofVL (reaching up to 25/100,000 in 0e6 year old children), ismore frequently complicated by concurrent infections suchas bronchopneumonia and diarrhoea.27 This variable clinicalexpression of leishmaniasis not only depends on the inocu-lated zymodeme and efficacy of the immune response, but isalso affected by environmental factors and the geneticconstitution of the host.28 Cryptic infections caused by vis-cerotropic Leishmania species are also common.29

The hepatosplenomegaly observed in our children withVL is a result of the accumulation of mononuclear phago-cytic cells which causes hyperplasia of reticulendothelialcells. Pancytopenia occurs secondary to bone marrow

involvement and hypersplenism. Concurrent viral infectionsand occasionally life-threatening bacterial sepsis may occursecondary to the associated immunosuppression.30

Untreated VL is fatal within 2e3 years. Persistence ofLeishmania is characteristic, and although none of thechildren in our case series relapsed, relapses can poten-tially occur up to 6e12 months after treatment.

Diagnosis of VL in our study was clinched by demon-stration of amastigotes by light microscopy of bone marrowsmears, with all children having intracellular amastigotes,known as Leishman-Donovan bodies (Fig. 1). Interestingly,an extracellular cyst-like structure (Fig. 1d) was noted onlyin children receiving antibiotics at the time of sampling.The well defined circular outline of the structure, as well asthe regular arrangement of the amastigotes within and thestaining characteristics of the interspersed material, makesit unlikely to represent cytoplasmic fragmentation ofa macrophage that may occur during smearing. This findinghas never been described previously and although its originis unexplained, could plausibly be a response to an adversemilieu created by the antibiotics. Electron microscopycould perhaps elucidate its morphology and significance.

Although not performed in our study, light microscopy ofsplenic or lymph node smears may alternatively be used fordiagnosing VL. We avoided splenic aspirates due to the

44 D. Pace et al.

potential risk of death from massive bleeding associatedwith thrombocytopenia, a frequent haematological mani-festation in Maltese children with VL. Lymph node smearswere not indicated as none of the described children hadlymphadenopathy. Leishmania may be cultured on a Novy-McNeal Nicolle medium but this is not done routinely inseveral countries due to the required expertise and cost.Despite lacking standardisation conserved sequences inminicircle kinetoplast DNA or in the small subunit rRNAgene of Leishmania may be detected rapidly by PCR onlymph node and bone marrow aspirates (Case 7), onperipheral blood31 or on urine.32

Some of our children with VL had anti-Leishmania anti-bodieswhich are useful diagnostically but arenot protective,probably due to the obligate intracellular nature of theparasite. Their detection by IF, ELISA orWestern blotmust becorrelated with clinical findings since false positives mayoccur in asymptomatic or resolved Leishmania infections andin other infectious diseases.33 Molecular mimicry of Leish-mania antigens, in addition to polyclonal B-cell activation,may result in the production of autoantibodies to ribonu-cleoproteins (Case3), rheumatoid factor and smoothmuscle,and may be responsible for a positive Coombs’ test (Case6).34,35 Autoantibodies, which are generally non-pathogenic,may play a yet undefined role in protection but can causediagnostic confusion with connective tissue disorders.36 Thedetection of serum anti-rK39 (an amino acid repeat that isconserved within the L. donovani complex) IgG37 or lowmolecular weight antigen (LMWA), thought to be a carbohy-drate antigen derived from amastigotes, in urine38 arealternative tests used for the rapid diagnosis of VL.

The Maltese children with LCL had ‘dry’ nodulo-ulcera-tive lesions (Fig. 2) typical of L. donovani complex disease.Diagnosis was based on the visualisation of amastigotes ondirect microscopy of skin smears (Fig. 4) or of punch/ellipseskin sections (Cases 9e11). Because of its benign presen-tation, LCL in children in Malta tends to be treated in thecommunity with cryotherapy without an attempt fora histopathological diagnosis and without notification. PCR

Figure 4 Cytology of slit skin smear showing extracellularand intracellular (within epithelioid histiocyte) amastigotes(Giemsa stain �600). Source: Pathology Department, Mater DeiHospital, Malta.

has improved the sensitivity of microscopy and can identifythe infecting species, but is costly and not done in Malta.39

The utility of serology is limited due to the low titres ofantibodies induced in LCL and therefore we do not performanti-Leishmania antibodies for children with LCL.39 Theleishmanin (Montenegro) skin-test, reflecting cell-mediatedimmunity, may be used but cannot distinguish between pastand present infection.39

Parenteral pentavalent antimonials have been thestandard treatment for both cutaneous and visceral diseasefor the last 50 years; however, the associated toxicity, longtreatment duration (20e28 days) and the development ofresistance from irregular compliance have led to the uti-lisation of alternative drugs.40 As in Malta, lipid formula-tions of amphotericin B are nowadays more commonly usedin Southern Europe due to their cost effectiveness whenadministered in short course regimens that results ina reduced hospital stay which offsets their high cost.41

Alternative agents such as the aminoglycoside paromomy-cin, is expected to provide a cheaper but efficaciousalternative to amphotericin B.42 Furthermore, miltefosine,the only oral formulation that has proven efficacy in chil-dren,43 may be used as outpatient therapy to treat VL,although non-compliance is a concern.

Although cutaneous lesions may be observed expectantlyas most would heal spontaneously within months, time toresolution varies between species and individuals. Intra-lesional antimony regimens administered between 1 and 3times weekly for 2e8 weeks (Case 11) are effective, butpainful. The local application of paromomycin ointment,44

cryotherapy45 or thermotherapy,46 are attractive optionsdevoid of systemic side effects, however, the success rate isvariable and species dependent. Prolonged and cosmeticallysignificant or multiple lesions may be treated with paren-teral antimonial compounds, amphotericin B, petamidineand oral miltefosine. Most of these treatment options havebeen poorly investigated in clinical trials and the need forproper research assessing the long term effects of suchregimens, particularly in children, is still there.47

Being an island there are several public health measuresthat can be implemented effectively to eradicate leish-maniasis fromMalta. Control measures on the importation ofdogs are already in place making it very unlikely for theintroduction of L. infantum from neighbouring endemicMediterranean countries. However, the current veterinarypractice of treating infected pet dogs with antimonialsseverely limits the control of canine leishmaniosis, since dogsare known to have high rates of relapse.48 In addition suchpractice promotes drug resistance48 and is very likely thereason for the treatment failures with antimonials seen insome of the children in our study. In the absence of a cullingprogram for infected dogs prospects for eradication ofleishmaniasis are bleak, although its implementation is likelyto be met with resistance from dog owners and animal rightsgroups.

Conversely, the promotion of deltamethrin-impregnateddog collars would be effective in preventing canine leish-maniosis and may result in a reduction in VL.49 Althoughvaccine prevention of leishmaniosis in dogs by means ofLeishmune�, the only licensed canine vaccine that iscurrently in use in Brazil is attractive, its impact on theepidemiology of zoonotic VL in humans is still unknown.50

Leishmaniasis in children in Malta 45

Vector control by indoor residual spraying of insecticidesis unpopular51 and only has a transient effect due to thepredominant exophagic and exophilic nature of mostphlebotomous sand flies, whilst outdoor spraying is inef-fective.19 Breeding ground destruction by removal of rubblewalls is illegal since these are part of the Maltese heritageand are protected. In Malta the wide distribution ofOpuntia trees found growing wildly or else being cultivatedfor their fruit from which a characteristic liqueur is alsomanufactured, makes their eradication challenging.

At present avoiding sand fly bites is the most practicaladvice that can be given to tourists visiting Malta in order toprevent leishmaniasis. The application of insect repellentson children who will be spending time outdoors after duskand before dawn, as is common during recreational activi-ties in summer in Malta, is encouraged. For those whoprefer to sleep with the windows open, insect screens arerecommended as these will help prevent getting bitten bysand flies indoors during sleep. Sleeping in storeys abovethe first floor is effective in avoiding sand fly biting as sandflies are poor fliers and can only hop a vertical distance of1 m.52 Immunisation against leishmaniasis is not possible atpresent since despite a century of research no effectivevaccine is available to protect against human leishmaniasis.

Conclusion

The manifestations of leishmaniasis described in this caseseries highlight the persistence of this parasitosis in Malta.The small contained size of Malta creates an opportunityfor eradication of leishmania which is, however, hinderedby the persistence of chronically infected dogs. Clinical,epidemiological and entomological studies of leishmaniasisin this country are needed. Eliminating the threat ofleishmaniasis would not only be beneficial for the Maltesepopulation but also for the tourist industry which is a majorsource of income for Malta. Unfortunately leishmaniasis isperceived as non-profitable by pharmaceutical companiesand failing industrial interest and investment in research,especially in developing countries, leishmaniasis willremain a neglected disease.

Author’s contribution statement

DP and TNW designed the study. DP, AG, AB, SAM, MJB andCV were involved in the acquisition, analysis and interpre-tation of the data. DP drafted the article which was criti-cally revised for important intellectual content by TNW,AG, AB, SAM, MJB and CV.

Conflict of interest

None declared.

Acknowledgements

The authors would like to thank Mr Peter Grech, Departmentfor Health Promotion and Disease Prevention, Malta and MsKathleen England, Department of Health Information, Malta

for providing the Maltese epidemiological data on leish-maniasis; and Dr Alex Aquilina, HSE Laboratories, Malta forproviding the data on positive leishmania serology in dogs.TNW is supported through a fellowship awarded by theWellcome Trust (076934).

References

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