Visceral leishmaniasis in an immunocompetent Hungarian adult patient

doi:10.4236/health.2011.31001

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Vol.3, No.1, 1-5 (2011) Health

doi:10.4236/health.2011.31001

Visceral leishmaniasis in an immunocompetent

Hungarian adult patient

Zoltán Péterfi1*, Zsuzsanna Nemes1, Szabolcs Vigvári1, Árpád Szomor1, László Kereskai2,

István Kucsera3, Balázs Tánczos4, Gábor Ternák1

11st Department of Internal Medicine, University of Pécs, Medical Centre, Pécs, Hungary

*Corresponding Author: [email protected]; The first two authors have equally contributed to the work

2 Department of Pathology, University of Pécs, Medical Centre, Pécs, Hungary

3 Department of Parasitology, National Centre for Epidemiology, Budapest, Hungary

4 Department of Parasitology and Zoology, Faculty of Vete rinary Science, Szent István University, Budapest, Hungary

Received 23 November 2010; revised 1 December 2010; accepted 3 December 2010

ABSTRACT

Background: V isceral leishmaniasis caused by L.

infantum Nicolle 1908 (Kinetoplastida, Trypano-

somatidae) is very rare in immunocompetent

adults. Clinical manifestation of the infection oc-

curs in children and immune-compromised pa-

tients associated with AIDS, haemopoietic ma-

lignancies or after renal, liver, and heart trans-

plantations in Europe. Hungary is regarded free

of leishmaniasis. Except for a single infection in

a small girl without a travel history, only a few

imported cases have been recorded among

Hungarians returning from endemic areas. Case

presentation: Visceral leishmaniasis was diag-

nosed in a 32 years old immumocompetent

Hungarian man who had spent his holidays

during the previous 3 years in Dalma tian territo-

ries (Dubrovnik, Makarska and Trogir) of Croatia.

He had two months history of fever, chills, and

night sweating associated with weight loss.

Physical examination showed mild hepatome-

galy and extreme splenomegaly. The protozoon

infection was confirmed by parasitological, se-

rological and molecular biological methods.

Clinical recovery of the patient was observed

af ter treatm ent with amphotericin B. Conclusion:

We want to highlight the effects of climate

changes and to call attention of health care

professionals in Central and Eastern European

countries to consider the possibility of leishma-

niasis as an emerging disease of tourists re-

turned from endemic regions.

Keywords: V isceral Leishmaniasis; Immunocompetent;

Human; Imported; Hungary; Amphotericin B

1. INTRODUCTION

Leishmaniasis is a group of the protozoan diseases,

transmitted by the bite of sandfly infected with Leish-

mania parasites [1]. The infection of humans appears

with multiple clinical manifestations including cutane-

ous (CL), mucocutaneous, diffuse and visceral (VL)

leishmaniasis. The latter is responsible for approx-

imately 59,000 deaths per year, a parasitic disease sur-

passed only by malar ia [2]. There are two types o f VL,

anthroponotic and zoonotic. Zoonotic VL (ZVL) is

wide spread and occurs in Latin America, Northern

Africa, Southern Europe and in areas of the Middle

East and Asia [2,3].

Two forms of leishmaniasis (VL and CL) present in

Europe are caused by Leishmania infantum [4]. While

cases of cutaneous leishmaniosis were reported in France,

Italy and Spain, ZVL is endemic in all countries border-

ing the Me diterranean Sea [5].

The reservoirs of the pathogen can be several wild and

domestic canid and rodent species but domestic dogs are

considered the main reservoir of L. infantum playing a

key role a s t he sourc e of human infection. Indeed, there is

a clear association between a high rate of infection in

dogs and a n i ncreased risk of human disease [1,2].

Geographically, the distribution of leishmaniasis is li-

mited by the distribution of its vector sandfly species.

The sandfly vectors are mainly active during the night,

and therefore the highest risk for contracting the infective

stages of the parasite from sandfly bites is between dusk

and dawn [6]. Five haematophagous sandfly species of

the subgenus Larroussius belonging to Phlebotomus ge-

nus (Diptera, Psychodidae) are known in Europe as the

vectors of L. infantum. Phlebotomus ariasi and P. perni-

ciosus are present in the western Mediterraneum from the

Iberian to the Italian peninsula, while P. perfiliewi, P.

neglectus and P. tobbi are distributed from Italy across

Z. Péterfi et al. / Health 3 (2011) 1-5

Balkan to Turkey [4,7,8]. The presence of these vector

species is essential for the endemic transmission of the

parasite among dogs and humans [9]. Another sandfly

species, P. mascitti is mentioned as a suspected vector

species of the parasite which is widely distributed in Eu-

rope with a northern limit in Belgium [6].

Our knowledge of the geographical distribution of

phlebotomine sandflies in Hungary is incomplete due to

the lack of systematic monitoring. According to histori-

cal records a few dozen specimens of P. macedonicus

(recently known as P. perfiliewi) wer e caught a t the sou-

theastern part of the country in the early 1930s [10].

During the recent surveys in the EU FP6 EDEN project

small numbers of two sandfly vectors (P. neglectus and P.

perfiliewi) were trapped close to the Croatian and Ser-

bian borders of the country (Farkas, personal communi-

cation).

In Europe, human VL is considered to be a rare dis-

ease, although its incidence increased significantly in the

region during the 1990s [6], about 700 autochthonous

cases have been reported each year from southern coun-

tries of Europe [3, 11]. Asymptomatic Leishmania infec-

tion of humans is more frequent than clinically apparent

form. Once the clinical illness begins the disease rapidly

progress and patients die due to VL unless treated. The

factors that determine the progress of VL have not yet

been completely identified, but a Leishmania specific

cellular immune response seems to play a fundamental

role in the control of infection [12 ]. After an incubation

period of 2-8 months (range: from 10 days to longer than

2 years), the patient develops pyrexia, wasting, and hepa-

tosplenomegaly which may become excessive. Amasti-

gotes of L. infantum disseminate throughout the reticu-

loendothelial system causing hyperplasia of phagocytic

cells in spleen, liver, bone marrow and sometimes l y mp h

nodes. In endemic European areas most patients are

children [13].

A historical review on human leishmaniasis in Croatia

documents the presence of stable disease foci in coastal

and insular territories of central and southern Dalmatia

since the beginning of the 20th century with a substantial

increase in incidence from the 1990s [14]. In contrast to

the endemic occurrence of the disease in the neighbor

Balkanic countries Hungary is regarded to be free of

leishmaniasis, only imported cases have been reported.

Over 30 cases are known that were imported from the

hyperendemic countries of the Middle East [15] and only

a single human case of ZVL has been diagnosed by

identification of amastigotes from the iliac crest biopsy

in Hungary that originated from the neighboring Croatia

that is regarded hypoendemic for leishmaniasis [16].

2. CASE PRESENTATION

On the 4th November, 2009, a 32 years old man was

admitted to the Department of Internal Medicine, Uni-

versity of Pécs because of two months history of fever,

chills, and night sweating associated with weight loss.

The patient had no history of any kind of previous ill-

nesses. Laboratory results revealed severe pancytopenia:

white blood cell: 0.85 G /l with 24% monocytes, hemog-

lobin: 86 g/l and platelet: 50 G/l. These findings can be

signs of hematological disorders (such as myelodysplas-

tic diseases) or may indicate several infections (e.g.,

brucellosis, Q fever, leishmaniasis). The erythrocyte sedi-

mentation rate and C-reactive protein (135 mg/l) were

elevated but normal level of alanine aminotransferase

and creatinine were observed. The gamma globulin level

was beyond the measurable range (>40 g/l) with de-

creased albumin level and albumin-globulin ratio of 0.4.

Physical examination showed mild hepatomegaly and

extreme splenomegaly (the spleen reached the upper rim

of the pelvis). The patient’s general condition was rela-

tively good. The reason for referral was a suspicion of

malignant hematological disease but the bone marrow

trephine biopsy excluded malignancy. The patient had

never received any transfusions or injections of blood or

blood products before the onset of his illness.

Detailed medical history revealed that the patient

spent his holidays during the previous 3 years in Dalma-

tian territories (Dubrovnik, Makarska and Trogir) of

Croatia. This raised the possibility of an imported “e xot-

ic” disease. Such imported disease which does not occur

in Hungary can be leishmaniasis although immunocom-

petent adults are affected very rarely. Examination of the

immune status of our recent patient excluded immuno-

suppression or HIV-1 co-infection. HIV- 1 test were

negative as well as the blood cultures, the Brucella ag-

glutination and Coxiella burnetti indirect immunofluo-

rescent assays.

The clinical diagnosis of visceral leishmaniasis was

confirmed by detection of amastigotes in bone-marrow

aspirate followed by serological and molecular assays.

Acute-phase serum sample was tested by Leishmania

Western-blot IgG qualitative Immunoblot Assay (Ldbio

Diagnostics, France), that showed well-defined bands

which are indicative for the presence of specific anti-

Leishmania IgG in the sample. The patient’s bone mar-

row aspirate and blood samp le were collected before and

after two week therapy, respectively. These samples

were studied with a L. donovani sl. kDNA minicircle

specific PCR assay [17]. DNA extracted with QIAamp

DNA Mini Kit (QIAgen, Hilden, Germany) was

processed in 25 µl final reaction-volume with RV1 (5’-

CTTTTCTGGTCCCGCGGGTAGG-3’) and RV2 (5’-

CCACCTGGCCTATTTTACACCA-3’) primers (25 pmole

both) and the PCR Core System I. (Promega, Madison,

WI, USA) to amplify an approximately 145 bp long re-

gion of the parasites DNA. Final concentrations

Z. Péterfi et al. / Health 3 (2011) 1-5

Figure 1. Fragments amplified in the L. dono-

vani sl. kDNA minicircle specific PCR assay.

BM: DNA extracted from bone marrow; BL:

DNA extracted from blood sample; N: negative

control; P: positive control; M: molecular weight

marker.

of the reagents were 1x for GoTaq Green buffer, 2 mM

for MgCl2 and 0.2 mM for each nucleotide, while 1.25

U of GoTaq DNA polymerase and 5 µl of DNA extract

were administered. Amplification was performed on a

Tpersonal 48 thermal cycler (Biometra, Göttingen, Ger-

many). An initial denaturation step for 4 minutes at 94 ˚C,

was followed by 40 cycles of 30 s 94 ˚C denaturation, 30

s 59˚C anellation and 30 s 72˚C elongation. A final ex-

tension at 72˚C for 10 minutes was followed by the hold

step at 4˚C. Products of amplification were visualized in

ethidium bromide stained 1.5% agarose gel. The PCR

assay proved the presence of Leishmania kDNA in the

patient´s bone marrow, but blood sample was negative

probably due to the efficacy of the treatment (Figu re 1).

Amphotericin B of total dose of 20 mg/body weight

was used conform to international recommendations.

After second day of treatment the patient become free of

fever, the leukocyte and platelet count continuously in-

creased reaching 3.63 G/l and 130 G/l respectively.

During treatment only temporary mild creatinine eleva-

tion (156 μmol/l) were observed, which normalized after

hydration. The patient’s spleen size decreased gradually;

on the second week of treatment declining half of the

original size whilst normalize at the end of treatment. In

the follow-up period no relapses were observed.

3. DISCUSSION

Classic ZVL in immunocompetent individuals in

Mediterranean countries is found mainly among children,

although since 1989 an increase in the number of cases

among adults has been observed in south-western Euro-

pean countries mostly (20-70%) due to Leishmania and

HIV-1 co-infectio ns. After a peak in 1990s, the incidence

of co-infections d ecreased notably due to introdu ction of

highly active antiretroviral therapy for AIDS. Even in

those areas as well where leishmaniasis is widespread

the incidences of ZVL among immunocompetent adults

were extremely rare [1,11 ,13,18,19].

Human VL has known to occur in Croatia since 1911,

although the first well-documented, microscopically

confirmed case was reported only in 1930 in the Du

brovnik area [14]. From 1931 through 1957 an average

of 15 cases were recorded yearly in several Dalmatian

territories. Starting from the 1960s, only sporadic cases

of both VL and CL were identified in central and south-

ern Dalmatia [14]. However, a substantial increase was

recorded in VL cases during the recent (1991-1995) w ar

in Croatia and in the post-war period in that region the

most of those cases (64.3%) were found in children be-

low 10 years [14].

Except for a single infection in a small girl without a

travel history [20], only imported human cases have

been recorded in Hungary. Várnai et al. [10] reported 31

cases of cutaneous or visceral leishmaniasis (CL, VL)

among Hungarians returning from endemic areas, and

Fried et al. [16] diagnosed VL in a Hungarian woman

who had spent her holidays in Dalmatia.

In the past two decades VL incidence rates without

HIV-1 co-infection have increased in Italy and France

(including a four-fold increase in Alpes-Maritimes), and

new endemic areas have been detected where no pre-

vious autochthonous cases had been reported (e.g. in

northern Italy, North Croatia, Switzerland and Germany)

[11,21]. The distribution of VL in Europe is significantly

more restricted than the distribution of the sandfly vec-

tors. The transmission of the disease agent within the

range of the vectors depends on vector abundance, vec-

tor survival, vector biting rate (i.e. gonotrophic cycle),

the extrinsic incubation period, and the length of the

transmission season. Each of these parameters is climate

dependent [6]. Climatic changes are likely to extend

northwards the range reached by the sandfly vectors. In

currently endemic areas, higher seasonal temperatures

would lead to prolonged activity periods and shorter

diapauses periods. This could result in an increased

number of sandfly generations per year. In addition,

higher temperatures may accelerate the maturation of the

protozoal parasite, thereby increasing the risk of infec-

tion [6,7,21]. Thus, climate-induced changes may in-

crease the risk of the emergence of new diseases includ-

ing leishmaniasis in Central and Western Europe [22,23].

Imported infected dogs can also contribute to the emer-

gence of leishmaniasis in new locations.

Whereas Hungary is regarded as free of leishmaniasis

the tourist traffic towards to the leishmania endemic re-

gions of Croatia is increasing year by year. So the

chance of appearance of new imported cases can be tak-

en into account. To prevent this greater emphasis should

be placed in education of travellers with a focus on the

available prevention methods.

Z. Péterfi et al. / Health 3 (2011) 1-5

4. CONCLUSIONS

Zoonotic VL is a re-emerging disease in the Mediter-

ranean area primarily due to climate induced changes.

Classic clinical pictures of the disease in immunocom-

petent individuals are found mainly among children.

Adults are affected very rare. Nevertheless as our case

suggests the occurrence of ZVL should be also consi-

dered in patients presenting with fever of unknown ori-

gin, hepatosplenomegaly, and pancytopenia who re-

turned from endemic areas.

5. ACKNOWLEDGMENT

The authors acknowledge the support provided by Prof. Róbert Far-

kas, Eszter Barna, Gábor Kovács and Zsofia Feiszt.

The molecular work was funded by EU grant GOCE-2003-010284

EDEN (Emerging Diseases in a changing European eNvironment)

and is catalogued by the EDEN Steering Committee as EDEN0236

(www.edenfp6project.net). The contents of this publication are the

sole responsibility of the authors and do not necessarily reflect the

views of the European Commission.

This work was supported by PTE ÁOK-KK grant No: 2010/10-23.

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