The emergence of Leishmania major and Leishmania donovani
in southern Turkey
Ismail S. Koltasa,*, Fadime Eroglua, Derya Alabazband Soner Uzunc
aDepartment of Parasitology, Faculty of Medicine, University of Cukurova, 01330 Balcali, Saricam, Adana, Turkey;bDepartment of
Pediatrics, Faculty of Medicine, University of Cukurova, 01330 Balcali, Saricam, Adana, Turkey;cDepartment of Dermatology, Faculty of
Medicine, University of Akdeniz, 07070 Konyaalti, Antalya, Turkey
*Corresponding author: Tel: +90 535 3059393; E-mail: email@example.com
Received 10 September 2013; revised 10 December 2013; accepted 10 December 2013
Background: In southern Turkey, Leishmania tropica and L. infantum are both the causative agents of cutaneous
leishmaniasis (CL) and visceral leishmaniasis (VL), respectively. However, L. majorand L. donovani were known to
exist after the influx of Syrian refugees.
Methods: Between the years of July 2003 and July 2013, a total of 167 smears and 113 bone marrow samples
Results: One hundred and seven 64% (107/167) smears and 56% (63/113) bone marrow samples were positive
for leishmaniasis according to the real-time PCR. Three different Leishmania species were found in the 107 CL
cases by real-time PCR: 42% (45/107) L. tropica, 36.5% (39/107) L. infantum and 21.5% (23/107) L. major. In
addition, three different Leishmania species were identified in the 63 VL cases: 60.3% (38/63) L. infantum,
30.2% (19/63) L. donovani and 9.5% (6/63) L. tropica using real-time PCR. The results of real-time PCR were con-
firmed with Leishmania ITS1 DNA sequencing.
Conclusions: This study revealed that in southern Turkey, L. majorand L. donovani were the aetiological agents of
CL and VL, respectively. It was assumed that emergence of L. major and L. donovani was due to influx of Syrian
refugees, as well as the effects of global warming.
Keywords: DNA Sequencing, Leishmania donovani, Leishmania major, Real-Time PCR, Turkey
mination of the clinical features of the disease, its follow-up and
the application of the optimal treatment in patients.1
Conventional laboratory methods for diagnosing leishmaniasis
are not adequate for the discrimination of species-specificity
due to the morphological similarity of different Leishmania spe-
cies.2Currently, the use of real-time PCR and DNA sequencing
are recommended as the most sensitive and convenient assays,
providing highly sensitive detection and quantification of
Leishmania species.3These methods can be applied for the pur-
pose of diagnosis, treatment efficacy studies, epidemiological
studies and vaccine trials.3,4
Cutaneous leishmaniasis (CL) and visceral leishmaniasis (VL) are
important public health problems in southern Turkey,5with CL
caused by L. tropica and VL caused by L. infantum.6,7Recent studies
have reported the presence of L. infantum in CL cases without a VL
therefore, aimed to identify the Leishmania species in southern
Turkey and investigated the effects of the influx of Syrian refugees
and global warming on the distribution of these species.
Materials and methods
Patients and ethics
Smear samples were taken from 167 CL-suspected cases in the
Department of Dermatology, Faculty of Medicine, Cukurova
University, Turkey. Bone marrow samples were taken from 113
VL-suspected cases in the Department of Paediatric Infectious
Diseases, Faculty of Medicine, Cukurova University, Turkey. CL
cases originated from Adana 49.1% (82/167) and other neigh-
bouring cities such as Hatay 18.5% (31/167), S ¸anlıurfa 14.4%
(24/167), Gaziantep 9.0% (15/167), Kahramanmaras ¸ 4.8%
(8/167) and Diyarbakır 4.2% (7/167) region. In addition, VL
cases came from Adana 54.9% (62/113) and other neighbouring
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cities such as Hatay 18.5% (21/113), S ¸anlıurfa 12.4% (14/113),
Gaziantep6.2% (7/113), Diyarbakır
Kahramanmaras ¸ 3.5% (4/113) region. Approval for the study
was obtained from the Ethics Committee of the Faculty of
Medicine, Cukurova University, Turkey.
Leishmania strains and DNA Extraction
The different Leishmania strains L. donovani (American Type
Culture Collection [ATCC], 30030D), L. infantum (ATCC, 501340),
L. major (ATCC, PRA-309D) and L. tropica (ATCC, 30012D) were
used as reference strains. DNA extraction was performed on all
samples using the QIAampDNA mini kit (Qiagen, Courtaboeuf,
France) according to the manufacturer’s instructions.
Genus-specific real-time PCR Assay
All samples (167 CL and 113 VL) were analysed using the genus-
specific real-time PCR assay. Genus-specific real-time PCR primers
were designed on the basis of the alignment of the Leishmania
minicircle kinetoplast (kDNA) gene region. The target gene kDNA
was present in copy numbers ranging from 10 000 to 20 000 in
the Leishmania species.
Genus-specific real-time PCR assay amplification was carried
out in a reaction mixture containing 10 ng of extracted DNA,
0.5 mM JW11 (5′CCT ATT TTA CAC CAA CCC CCA GT3′), 0.5 mM
JW12 (5′GGG TAG GGG CGT TCT GCG AAA3′) and 1X QuantiFast
SYBR Green PCR kit (Qiagen, Valencia, CA, USA) (10 mM Tris-HCl,
50 mM KCl, 2 mM MgCl2, 1 mM Rox dye, 2 U Taq polymerase and
200 mM each dNTP).12The reaction was brought to 20 mL total
volume with PCR grade water. The PCR was carried out using a
Rotor Gene RG-3000 (Corbett Research, San Francisco, CA, USA)
with a programme consisting of warming to 508C for 2 min, initial
denaturation at 958C for 1.5 min, followed by 35 cycles of
denaturation at 958C for 15 s, annealing at 658C for 30 s, exten-
sion at 728C for 30 s and a final extension at 728C for 10 min.
The fractional cycle number reflecting a positive PCR result is
called the cycle threshold (CT). The CTvalues were automatically
meters. The standard curves were also used to determine the
detection limit of the assays and to assess the linearity (R2) and
the efficiency (E) of the real-time PCR amplifications.
Species-specific real-time PCR Assay
The primers JW13 (5′ACT GGG GGT TGG TGT AAA ATA GG3′) and
JW14 (5′TTT CGC AGA ACG CCC CTA CCC3′), designed on the con-
served region of the Leishmania kDNA minicircle, were used to
allow the amplification of L. donovani, L. infantum, L. major and
The species-specific real-time PCR reaction consisted of a total
volume of 20 ml, containing 10 ng of extracted DNA, 0.5 mM pri-
mer mix (forward and reverse), 1X QuantiFast SYBR Green PCR
kit (Qiagen) (10 mM Tris-HCl, 50 mM KCl, 2 mM MgCl2, 1 mM Rox
dye, 2 U Taq polymerase, and 200 mM each dNTP) and 4 mL dis-
tilled water. Species-specific real-time PCR assay were carried
out in a Rotor-Gene RG-3000 (Corbett Research, San Francisco,
CA, USA). All programmes included a process of warming to
508C for 2 min, initial denaturation at 958C for 4 min, followed
by 35 cycles of denaturation at 958C for 10 s, annealing at 628C
for 10 s, extension at 728C for 10 s, and a final extension at
728C for 10 min. The melting programme consisted of one cycle
of 958C for 10 s, 678C for 20 s and heating at 988C. The transition
had a temperature transition rate of 18C/s and 0.18C, respectively.
The melting temperatures (Tm) were 87.5+0.38C for L. major,
88.5+0.48C for L. donovani, 89.3+0.28C for L. tropica and
90.5+0.28C for L. infantum, respectively.
All of the reactions were analysed using the software provided
with the instrument. The average CTvalues were determined and
the standard curves were calculated using the Rotor-Gene 6.1.93
Confirmation of real-time PCR results by sequencing
The results of real-time PCR were confirmed by the Leishmania
ITS1 DNA sequencing method. The PCR was carried out using
the LITSR-L5.8S primers.14PCR products were purified using a
SentroPure DNA purification kit (Sentromer DNA, Istanbul,
Turkey). They were sequenced with same combination of primers
using the BigDye Terminator V 3.1 cycle sequencing kit (Applied
Biosystems, Carlsbad CA, USA), as per the manufacturers’ proto-
col, on the 3730 DNA analyser (Applied Biosystems, California,
USA). The ITS1 PCR products of Leishmania isolate sequences
were found to be approximately 300 bp in length. The sequences
obtained were processed using GenBank and checked using basic
local alignment search tool (BLAST) analysis software (www.ncbi.
Results of genus-specific real-time PCR Assay
Form the 167 smear samples taken from suspected CL patients,
107/167 (64.1%) samples were found to be positive and 60/167
(35.9%) were negative, using genus-specific real-time PCR assay
(Table 1). The Leishmania parasite was detected in 63/113
(55.8%) of bone marrow samples while 50/113 (44.3%) bone
marrow samples were negative according to genus-specific real-
time PCR assay (Table 3).
The CTvalue of genus-specific real-time PCR assay was found
On average the R2value of the standard curve was .0.99 in all
real-time PCR assays.
Results of species-specific real-time PCR Assay
Three different Leishmania species were identified in 107 DNA
smear samples: 42.1% L. tropica (45/107), 36.5% L. infantum
(39/107) and 21.5% L. major (23/107). L. donovani was not
detected in CL patients (Table 2). In addition, three different
Leishmania species were found in 63 bone marrow DNA samples:
60% L. infantum (38/63), 30% L. donovani (19/63) and 10% L. tro-
pica (6/63). L. major was not observed in VL patients (Table 4).
Of the 23 CL cases with L. major, 14 cases (61%, 14/23) were
Syrian and 9 (39%, 9/23) were Turkish. Furthermore, of the 19 VL
19) Turkish. The study group did not have any Iranian, Iraqi and
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Table 1. The results of genus-specific real-time PCR assay in cutaneous leishmaniasis cases
Locality Positive, %Negative, % Total, %
Table 3. The results of genus-specific real-time PCR assay in visceral leishmaniasis cases
LocalityPositive, % Negative, % Total, %
Table 2. The results of species-specific real-time PCR assay in cutaneous leishmaniasis cases
Locality Leishmania tropica, %Leishmania major, %Leishmania infantum, %Leishmania donovani, %
Table 4. The results of species-specific real-time PCR assay in visceral leishmaniasis cases
Locality Leishmania tropica, %Leishmania major, % Leishmania infantum, %Leishmania donovani, %
10 (6/63)30 (19/63)
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The CTvalues of all species-specific real-time PCR assays were
found to be between 21.2 and 39.1 with a median threshold of 32
cycles. On average the R2value of the standard curve was .0.99
in all real-time PCR assays.
Results of ITS1 DNA sequencing
Each clinical specimen and ATCC reference strain of Leishmania
species (L. donovani, L.infantum, L.tropica andL. major) was iden-
tical to the sequence of Leishmania species reported in GenBank.
As compared to the previous records in GenBank, the similarity
between sequences of Leishmania isolates from CL cases and
Leishmania strains in GenBank was 97–99% (L. tropica 98%,
L. infantum 99% and L. major 97%). There was a 2% nucleotide
difference between L. tropica and JN860722 in 200–230 bp,
while L. infantum exhibited a 1% nucleotide difference with
GQ444144 in 240–260 bp. There was a 3% nucleotide difference
between L. major and AY550178 in 180–200 bp.
The LeishmaniaisolatesfromVL revealeda 97–100% similarity
with Leishmania strains in GenBank (L. infantum 98%, L. donovani
97% and L. tropica 99%). There was a 2% nucleotide difference
between L. infantum and HQ535858 in 200 bp. L. donovani
showed a 3% nucleotide difference with AJ249615 and there
was a 1% difference between L. tropica and HM004586 in 160 bp.
The causative agents of CL (L. major) and VL (L. donovani) are
known to be endemic in countries bordering Turkey such as Iraq,
Iran and Syria.15,16L. tropica, L. infantum and L. major have been
detected however, L. donovani has not been previously identified
identify any signs of L. major in southern Turkey.9However, in this
current study, we were able to isolate L. major from CL cases in
We determined that there are two possible explanations for
our recent findings. Firstly, Turkey represents a crossroads
between the European and Asian continents and different eco-
logical and climatic conditions exist. Global warming has had a
severe effect on most species and on Phlebotomus in particu-
lar.18,19Secondly, many Syrian refugees have flocked to Turkey
since March 2011, with numbers reaching 506 532 in October
2013. Refugee camps have been set up in the southern cities of
Hatay, S ¸anlıurfa, Gaziantep, Diyarbakır, Kahramanmaras ¸ and
Adana province. Some refugees are however living outside the
camps. Health care for the refugees has been provided free of
charge by health institutions.20,21Many refugees have been suf-
fering from poor health caused by a variety of different sources.
They have also come from areas with poor sanitary conditions
and many health hazards, before settling in Turkey.20
Demographic data revealed that there were no Iranian, Iraqi
and Afghani among those who immigrated to Turkey, despite
southern Turkey being on the route for immigrants coming from
Iran, Iraq and Afghanistan on their way to Europe. Thus, we
believe that the incidence of L. major and L. donovani might be
associated with the Syrian refugee population, since Turkish
patients infected with both species were found to be living near
the refugee camps.
The majority of isolates identified were L. infantum and L. tro-
pica, which have been known to cause zoonotic VL and
anthroponotic CL in western and southeastern Turkey, respect-
ively.11The findings in this study revealed that L. major was
one of the causative agents of zoonotic CL and L. donovani
was one of the causative agents of anthroponotic VL in southern
Turkey. The main vectors of leishmaniasis are several
Phlebotomus species and the main animal reservoir host is the
dog.22P. papatasi and P. duboscqi have been identified as the
main vector of L. major and P. orientalis is known as the vector
of L. donovani. P. duboscqi and P. orientalis have not been
reported, whereas P. papatasi has been detected in Turkey.22,23
P. papatasi has been reported to be the main vector of
L. major, so it is possible that it is responsible for the transmission
of L. major from Syrian refugees to Turkish patients. The data on
causative agents, vectors and reservoirs of diseases are not
regularly updated and verified; therefore information available
in the past has changed in recent years due to environmental
modifications, global warming and population movements.22
P. duboscqi and P. orientalis are likely to have become vectors
of transmission due to the above mentioned environmental
modifications. In addition, molecular studies and the sequen-
cing of the Leishmania genome have increasingly suggested
new approaches regarding the treatment, prognosis and control
of the disease.24It is vital that this research is continued for
improved diagnosis and the control of the disease.25
Furthermore, in the present study, L. infantum was detected
in CLwithout VL history, while L. tropica was identified in VLwith-
out CL history, using species-specific real-time PCR and ITS1 DNA
sequencing methods. CL caused by L. infantum, and L. tropica as
a causative agent of VL in Iran and Turkey has recently been
reported.10,11Toz et al. reported 52.5% (117/223) L. infantum
in CL cases.11Our findings appear to confirm these findings as
we showed that 36.5% (39/107) L. infantum caused CL in
Turkey. In addition, Alborzi et al. identified only one (1/64) L. tro-
pica among 64 bone marrow/spleen samples while Toz et al.
found 7% (6/92) L. tropica among 92 human and canine VL
cases.10,11They reported a rare causative agent of VL while we
found 9.5% (6/63) L. tropica among 63 VL cases using species-
specific real-time PCR and ITS1 DNA sequencing. This percentage
detection is higher than those of the other studies, which sug-
gests that L. tropica isthe main causativeagent of VL in southern
The main limitation of the study is related to the methodology
usedinthatwecouldnotperforma zymodemanalysisfor strains.
The other limitation is that we did not investigate the relation
between Leishmania species and the clinical characteristics of
Using species-specific real-time PCR and ITS1 DNA sequencing,
prevalent in southern Turkey. This result indicates that Leishmania
species show a cosmopolitan distribution in this region. In add-
ition the results of the studyon the diagnosis of CLand VLcontrib-
ute to ourcurrent understandingof leishmaniasis epidemiologyin
southern Turkey. However, future studies should be carried out to
further explore these issues.
Authors’ contributions: ISK conceived the study, analysed the data and
wrote the sections on context; FE assisted in the study conception and
I. S. Koltas et al.
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analysed the samples using molecular methods; DA took bone marrow Download full-text
from VL-suspected patients and contributed to the writing of the paper.
SU took smear samples from CL-suspected patients. All authors read
and approved the final paper. ISK and FE are guarantors of the paper.
Funding: Funding was provided by the Cukurova University Research Grant
Competing interesting: None declared.
Ethical approval: The Ethical Committee of the Medicine Faculty of
Cukurova University gave approval for this study.
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