(VL) cases has recently been reported in Bologna Province in northern Italy. Over six months from November 2012 to May 2013, 14 cases occurred, whereas the average number of cases per year was 2.6 (range: 0–8) in 2008 to 2012. VL was diagnosed in a median of 40 days (range: 15–120) from disease onset. This delay in diagnosis shows the need for heightened awareness of clinicians for autochthonous VL in Europe. From November 2012 to May 2013, public health authorities, microbiologists and clinicians in Bologna Province, northern Italy, noted an upsurge in human cases of visceral leishmaniasis. During these six months, 14 cases were notified, an over five-fold increase compared with the annual average of 2.

A C Finarelli

C Salvadori

F Melchionda

G A Gentilomi

L Attard

R Cagarelli

R Rangoni

R Tigani

S Varani (stefania. Varani@unibo. It

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1 www.eurosurveillance.org
Rapid communications
Ongoing outbreak of visceral leishmaniasis in Bologna 
Province, Italy, November 2012 to May 2013
S Varani (stefania.varani@unibo.it)1, R Cagarelli2, F Melchionda3, L Attard4, C Salvadori4, A C Finarelli2, G A Gentilomi1, R Tigani1, 
R Rangoni5, R Todeschini6, A Scalone7, T Di Muccio7, M Gramiccia7, L Gradoni7, P Viale4, M P Landini1
1.  Unit of Microbiology, Regional Reference Center for Microbiological Emergencies (CRREM), St. Orsola Malpighi Hospital, 
University of Bologna, Bologna, Italy
2. Public Health Authority Emilia-Romagna, Bologna, Italy
3. Department of Pediatric Hematology and Oncology, St. Orsola Malpighi University Hospital, Bologna, Italy
4. Infectious Disease Unit, St. Orsola Malpighi Hospital, University of Bologna, Italy
5.  Public Health Department of Imola, Bologna, Italy
6. Public Health Department of Casalecchio di Reno, Bologna, Italy
7.  Unit of Vector-Borne Diseases and International Health, Istituto Superiore di Sanità, Rome, Italy
Citation style for this article: 
Varani S, Cagarelli R, Melchionda F, Attard L, Salvadori C, Finarelli AC, Gentilomi GA, Tigani R, Rangoni R, Todeschini R, Scalone A, Di Muccio T, Gramiccia 
M, Gradoni L, Viale P, Landini MP. Ongoing outbreak of visceral leishmaniasis in Bologna Province, Italy, November 2012 to May 2013. Euro Surveill. 
2013;18(29):pii=20530. Available online: http://www.eurosurveillance.org/ViewArticle.aspx?ArticleId=20530
Article submitted on 20 June 2013 / published on 18 July 2013
An increased number of autochthonous visceral leish-
maniasis (VL) cases has recently been reported in 
Bologna Province in northern Italy. Over six months 
from November 2012 to May 2013, 14 cases occurred, 
whereas the average number of cases per year was 2.6 
(range: 0–8) in 2008 to 2012. VL was diagnosed in a 
median of 40 days (range: 15–120) from disease onset. 
This delay in diagnosis shows the need for height-
ened awareness of clinicians for autochthonous VL in 
Europe.
From November 2012 to May 2013, public health 
authorities, microbiologists and clinicians in Bologna 
Province, northern Italy, noted an upsurge in human 
cases of visceral leishmaniasis. During these six 
months, 14 cases were notified, an over five-fold 
increase compared with the annual average of 2.6 
cases (range: 0–8) from 2008 to 2012. Here, we report 
preliminary epidemiological, microbiological and clini-
cal findings.
Background
Visceral leishmaniasis (VL) is a severe disease pri-
marily affecting the host’s reticuloendothelial system 
and is caused by parasitic protozoans belonging to 
the  Leishmania donovani complex. VL is endemic in 
Mediterranean Europe, where the disease is caused by 
L. infantum. Transmission is mainly zoonotic and occurs 
via the bite of a phlebotomine sandfly species of the 
genus Phlebotomus [1,2]. In Italy, the Tyrrhenian lit-
toral, the southern peninsular regions and the islands 
have been considered classical endemic zones for VL, 
whereas in continental northern regions, VL has mainly 
affected human immunodeficiency virus (HIV)-positive 
patients. Since the early 1990s, however, a north-
ward spread of the disease to previously non-endemic 
Italian regions has been observed. These regions also 
exhibited a progressive decrease in HIV co-infection 
rates [3]. A recent survey conducted in a continental 
area of north-western Italy, which was previously con-
sidered to be non-endemic, detected anti-leishmanial 
antibodies in 7.4% of healthy adults; for half of the 
seropositives, ongoing infection was confirmed by PCR 
analysis [4]. Currently, the occurrence of both asymp-
tomatic and symptomatic leishmaniasis seems to be 
underestimated in Italy. 
Outbreak description 
In Italy, laboratory-confirmed cases of VL are reported 
by local public health departments to the regional 
authorities, which report cases to the Ministry of 
Health. The case definition for VL in Italy is based on 
the World Health Organization (WHO) case definition 
Figure 1
Epidemic curve of human cases of visceral leishmaniasis, 
Bologna Province, northern Italy, November 2012–May 
2013 (n=14)
0
1
2
3
4
5
6
Nov Dec Jan Feb Mar Apr
2012 2013
N
u
m
b
e
r
 
o
f
 
c
a
s
e
s
 
Date (disease onset)
 2 www.eurosurveillance.org
and includes positive serology (indirect immunofluo-
rescence antibody test (IFAT), ELISA, rK39-based immu-
nochromatographic test (IC), direct agglutination test) 
and/or parasitology (microscopy, culture or PCR) for 
patients with suspected clinical symptoms [5].
From November 2012 to May 2013, 14 new cases of 
VL occurred in Bologna Province in northern Italy 
(Figure 1). Most patients resided in hilly, rural areas. 
Municipalities and their altitudes are reported in Figure 
2.
The age range of patients was five months to 83 years, 
four cases were aged 18 months or younger, five were 
between 48 and 60 years-old, and five were between 
62 and 83 years-old. The majority, 11 patients, were 
male. One patient was HIV-positive. Overall, five 
patients had known risk factors for VL, i.e. being under 
two years-old, (n=4) or being HIV-positive (n=1). All 
patients had symptoms compatible with VL, includ-
ing a fever of unknown origin (n=13), mild to moderate 
anaemia (n=12), leukopenia (n=12), thrombocytopenia 
(n=10), hepatomegaly (n=7), splenomegaly (n=14) and 
weight loss (n=7). Interestingly, four of 14 VL cases 
presented with haemophagocytic syndrome (HS), a 
systemic hyperinflammatory disorder with severe dys-
function of immune homeostasis that may be second-
ary to VL [6,7]. 
VL was diagnosed in a median time of 40 days from 
disease onset (range: 15–120) and was supported by 
parasitological and serological means. 
Laboratory investigations
Bone marrow aspirate was performed in 12 cases, but 
smear was available for microscopic diagnosis only in 
10 cases. The detection of amastigotes was positive 
in three of 10 cases investigated, and the culture was 
negative in all nine tested cases (Table), suggesting a 
low parasite load in the bone marrow [8]. As expected 
[9], molecular methods enhanced parasite detection in 
bone marrow samples. Leishmanial DNA was detected 
by nested PCR and/or real-time PCR in seven of 10 
patients and in the peripheral blood of an additional 
patient who was HIV-positive; thus, molecular methods 
were positive in eight of 13 VL cases (not performed in 
one case). 
Serology performed using an indirect haemagglutina-
tion assay (IHAT) was negative in all eight cases tested, 
whereas the rk39-based IC [10] and IFAT were positive 
in all tested patients (10 of 10 and 13 of 13, respectively, 
see Table). Thus, serodiagnosis by IFAT or IC in patients 
with suspected symptoms was fundamental. 
Indeed, in four of five cases in whom a diagnosis of 
VL was posited based on only suggestive symptoms 
and serological tests, the response to anti-leishmanial 
treatment (liposomal amphotericin B, 10 mg/kg intra-
venous, single dose) was appropriate, suggesting 
that our diagnostic approach was correct. One case 
resolved symptoms without treatment and is currently 
under clinical and laboratory evaluation. 
Discussion
The risk of the emergence or resurgence of several 
exotic vector-borne pathogens in Europe, including chi-
kungunya and dengue virus, has become a hot topic 
over the past decade, whereas other infections, such 
as leishmaniasis, have been neglected [11]. In fact, cli-
nicians and microbiologists are often ill informed on 
the prevalence and symptoms of, and detection meth-
ods for VL, which may lead to initial misdiagnosis and 
a delay in diagnosis and treatment.
According to the epidemic curve (Figure 1), most cases 
of the ongoing outbreak in Bologna Province had 
disease onset in the winter months, indicating that 
patients were probably infected during summer and 
autumn 2012. Furthermore, half of the cases were diag-
nosed more than 40 days from disease onset and one of 
these cases was diagnosed 120 days after initial symp-
toms. This indicates a frequent delay in identifying the 
Figure 2
Geographical location of human cases of visceral 
leishmaniasis, Bologna Province, northern Italy, 
November 2012–May 2013 (n=14)
m: meters above sea level
! (
! (
! (
! (
! (
! (
! (
! (
! (
! (
! (
Bologna - 54 m
Imola - 47 m
Casalfiumanese - 125 m
Monterenzio - 207 m
Pianoro - 200 m
Sasso Marconi - 128 m
Monte San Pietro - 112 m
Monteveglio - 114 m
Castello di Serravalle - 182 m
Savigno - 259 m
Grizzana Morandi - 547 m
±
01 02 03 0 5
Kilometers
! ( 1 case
! ( 2 cases3 www.eurosurveillance.org
parasitic infection. One third of the identified cases 
were revealed as having HS, and one child was errone-
ously diagnosed as having familial haemophagocytic 
lymphohistiocytosis (FHL). Symptoms for FHL and HS 
due to VL may overlap, and the differential diagnosis 
can be difficult [7, 12]. Thus, awareness of the increas-
ing incidence of VL in areas previously considered to 
be at low risk is fundamental to avoid misdiagnosis, 
especially in infants in whom HS might be confused 
with FHL. 
L. infantum is considered to be autochthonous in 
Bologna Province and P. perfiliewi is the predominant 
vector in this area [13]. In 1971–72, an outbreak of VL 
occurred in Bologna Province that affected 60 individu-
als, with 13 deaths [14]. Afterwards, the area remained 
endemic, with a low prevalence and an annual mean 
of 2.6 reported cases (range: 0–8 years) for the years 
2008 to 2012 (R. Cagarelli, personal communication, 
May 2013). The reasons for the recent upsurge of VL 
cases in Bologna Province are unknown. Theoretically, 
current global warming in the Mediterranean area [15] 
may enhance leishmaniasis distribution due to the 
effect of temperature on parasite development and 
vector spread. However, according to WHO, there is no 
clear evidence indicating that sandfly and VL distribu-
tion in Europe have changed in response to climate 
change [2]. 
In conclusion, the increase in human VL cases in an 
area of northern Italy raises important public health 
concerns. Firstly, there is an urgent need to expand 
the existing control measures for canine leishmania-
sis [13]. Secondly, healthcare professionals need to be 
informed of the upsurge in VL in the area to proceed 
with appropriate parasitological and serological tests 
in suspected cases, to promptly identify and treat 
cases of VL. Finally, the public needs to be aware of the 
potential exposure to sandfly bites in areas in which 
the parasite circulates, and to be educated in the use 
of appropriate preventive measures, such as mechani-
cal and chemical repellents.
Acknowledgements 
This study was supported by the Emilia-Romagna Region 
(Laboratory P3 funds) and by the University of Bologna (RFO 
funds). 
Conflict of interest
None declared.
Authors’ contributions
Designed the study: SV and MPL. Wrote the first draft: SV. 
Collected, synthesised and analysed the data: RC, ACF, LA, 
CS, FM, MG, AS, TDM, RT, GAG, RR and RT. Interpreted the 
results critically and revised the article to ensure important 
intellectual content: PV, LG and MPL. All authors read and 
approved the final manuscript.
Table 
Parasitological and serological findings in visceral leishmaniasis cases, Bologna Province, Italy, November 2012–May 2013 
(n=14)
Case number Time to diagnosis Parasitological findings Serological findings
1 78 days BM: microscopy - , PCR+ IHAT-, IC+, IFAT+ (1:80)
2  27 days BM: microscopy- , culture-, PCR+ IHAT-, IC+, IFAT+ (1:160)
3  15 days BM: microscopy -, culture-, PCR- IHAT-, IC+, IFAT+ (1:160) 
4  42 days BM: microscopy - , culture-,PCR- IC+, IFAT+ (1:40)
5 40 days BM: microscopy+, culture-, PCR+ IHAT-, IFAT+ (1:80)
6 15 days BM: microscopy -culture-, PCR- IC+, IFAT+ (1:1,280)
7 80 days BM: microscopy -, culture-, PCR- IHAT-, IC+, IFAT+ (1:5,120)
8  40 days PB: PCR+ IHAT-, IC+, IFAT+ (1:10,240)
9 120 days PB: culture-, PCR- IHAT-, IC+, IFAT+ (1:5,120)
10  20 days BM: PCR+  IHAT-
11 30 days BM: microscopy +  IFAT+ (1:320)
12  25 days BM: microscopy - , PCR+ IFAT+ (1:1,280)
13 60 days BM: microscopy+, culture-, PCR+ IC+, IFAT+ (1:320)
14  50 days BM: culture- , PCR+ IC+, IFAT+ (1:640)
BM: bone marrow; PB: peripheral blood; IFAT: indirect immunofluorescence antibody test; IHAT: indirect haemagglutination test; IC: rk39-
based immunochromatographic test; -: negative; +: positive.4 www.eurosurveillance.org
References
1.  Gramiccia M, Gradoni L. The current status of zoonotic 
leishmaniases and approaches to disease control. Int J 
Parasitol. 2005;35(11-12):1169-80. http://dx.doi.org/10.1016/j.
ijpara.2005.07.001. PMid:16162348.  
2.  Ready PD. Leishmaniasis emergence in Europe. Euro 
Surveill. 2010;15(10):pii=19505. Available from: http://www.
eurosurveillance.org/ViewArticle.aspx?ArticleId=19505. 
PMid:20403308.  
3.  Maroli M, Rossi L, Baldelli R, Capelli G, Ferroglio E, Genchi 
C, et al. The northward spread of leishmaniasis in Italy: 
evidence from retrospective and ongoing studies on the 
canine reservoir and phlebotomine vectors. Trop Med Int 
Health. 2008;13(2):256-64. http://dx.doi.org/10.1111/j.1365-
3156.2007.01998.x. PMid:18304273.  
4.  Biglino A, Bolla C, Concialdi E, Trisciuoglio A, Romano A, 
Ferroglio E. Asymptomatic Leishmania infantum infection in 
an area of northwestern Italy (Piedmont region) where such 
infections are traditionally nonendemic. J Clin Microbiol. 
2010;48(1):131-6. http://dx.doi.org/10.1128/JCM.00416-09. 
PMid:19923480. PMCid:PMC2812267. 
5.  World health Organization (WHO). Control of the 
leishmaniases. In: WHO Technical Report Series. Geneva: WHO; 
2010. p. 175-6. Available from: http://whqlibdoc.who.int/trs/
WHO_TRS_949_eng.pdf 
6.  Henter JI, Horne A, Arico M, Egeler RM, Filipovich AH, Imashuku 
S, et al. HLH-2004: Diagnostic and therapeutic guidelines for 
hemophagocytic lymphohistiocytosis. Pediatr Blood Cancer. 
2007;48(2):124-31. http://dx.doi.org/10.1002/pbc.21039. 
PMid:16937360.  
7.  Rajagopala S, Dutta U, Chandra KS, Bhatia P, Varma N, 
Kochhar R. Visceral leishmaniasis associated hemophagocytic 
lymphohistiocytosis--case report and systematic review. 
J Infect. 2008;56(5):381-8. http://dx.doi.org/10.1016/j.
jinf.2008.02.013. PMid:18405976.  
8.  Chappuis F, Sundar S, Hailu A, Ghalib H, Rijal S, Peeling RW, et 
al. Visceral leishmaniasis: what are the needs for diagnosis, 
treatment and control? Nat Rev Microbiol. 2007;5(11):873-82. 
http://dx.doi.org/10.1038/nrmicro1748. PMid:17938629.  
9.  Antinori S, Calattini S, Longhi E, Bestetti G, Piolini R, Magni 
C, et al. Clinical use of polymerase chain reaction performed 
on peripheral blood and bone marrow samples for the 
diagnosis and monitoring of visceral leishmaniasis in HIV-
infected and HIV-uninfected patients: a single-center, 8-year 
experience in Italy and review of the literature. Clin Infect 
Dis. 2007;44(12):1602-10. http://dx.doi.org/10.1086/518167. 
PMid:17516404.  
10.  Brandonisio O, Fumarola L, Maggi P, Cavaliere R, Spinelli R, 
Pastore G. Evaluation of a rapid immunochromatographic test 
for serodiagnosis of visceral leishmaniasis. Eur J Clin Microbiol 
Infect Dis. 2002;21(6):461-4. http://dx.doi.org/10.1007/s10096-
002-0739-8. PMid:12111603.  
11.  Dujardin JC, Campino L, Canavate C, Dedet JP, Gradoni 
L, Soteriadou K, et al. Spread of vector-borne diseases 
and neglect of Leishmaniasis, Europe. Emerg Infect Dis. 
2008;14(7):1013-8. http://dx.doi.org/10.3201/eid1407.071589. 
PMid:18598618. PMCid:PMC2600355. 
12.  Singh G, Shabani-Rad MT, Vanderkooi OG, Vayalumkal JV, 
Kuhn SM, Guilcher GM, et al. Leishmania in HLH: a rare 
finding with significant treatment implications. J Pediatr 
Hematol Oncol. 2013;35(3):e127-9. http://dx.doi.org/10.1097/
MPH.0b013e318286d619. PMid:23511497  
13.  Baldelli R, Piva S, Salvatore D, Parigi M, Melloni O, Tamba 
M, et al. Canine leishmaniasis surveillance in a northern 
Italy kennel. Vet Parasitol. 2011;179(1-3):57-61. http://dx.doi.
org/10.1016/j.vetpar.2011.01.052. PMid:21349642.  
14. Pampiglione S, La Placa M, Schlick G. Studies on 
mediterranean Leishmaniasis. I. An outbreak of 
visceral leishmaniasis in Northern Italy. Trans R Soc 
Trop Med Hyg. 1974;68(5):349-59. http://dx.doi.
org/10.1016/0035-9203(74)90148-5 
15.  Voiland A. 2009: Second warmest year on record; end of 
warmest decade. Washington: National Aeronautics and 
Space Administration (NASA); Jan 2010. Available from: http://
climate.nasa.gov/news/249 

Rapid communications Ongoing outbreak of visceral leishmaniasis in Bologna

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Rapid communications Ongoing outbreak of visceral leishmaniasis in Bologna

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