Transactions of the Royal Society of Tropical Medicine and Hygiene Advance Access published January 20, 2014
ORIGINAL ARTICLE
Trans R Soc Trop Med Hyg doi:10.1093/trstmh/trt119
The emergence of Leishmania major and Leishmania donovani in southern Turkey Ismail S. Koltasa,*, Fadime Eroglua, Derya Alabazb and Soner Uzunc a
Department 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
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. major and 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 were taken from CL and VL-suspected cases, respectively. Samples were analysed through real-time PCR and ITS1 DNA sequencing. 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 confirmed with Leishmania ITS1 DNA sequencing. Conclusions: This study revealed that in southern Turkey, L. major and 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
Introduction The identification of Leishmania species is important for the determination 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 species.2 Currently, 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.3 These methods can be applied for the purpose 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,5 with CL caused by L. tropica and VL caused by L. infantum.6,7 Recent studies have reported the presence of L. infantum in CL cases without a VL history.8,9 There have also been reports of the presence of L. tropica in
VL cases without a CL history in both Iran and Turkey.10,11 This study, 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 neighbouring 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
# The Author 2014. Published by Oxford University Press on behalf of Royal Society of Tropical Medicine and Hygiene. All rights reserved. For permissions, please e-mail: [email protected].
1 of 5
Downloaded from http://trstmh.oxfordjournals.org/ at Belgorod State University on February 11, 2014
*Corresponding author: Tel: +90 535 3059393; E-mail: [email protected]
I. S. Koltas et al.
cities such as Hatay 18.5% (21/113), S¸anlıurfa 12.4% (14/113), Gaziantep 6.2% (7/113), Diyarbakır 4.5% (5/113) and 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
Genus-specific real-time PCR Assay All samples (167 CL and 113 VL) were analysed using the genusspecific 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).12 The 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, extension 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 CT values were automatically determined by the Rotor-Gene 6.1.93 software using default parameters. 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 conserved region of the Leishmania kDNA minicircle, were used to allow the amplification of L. donovani, L. infantum, L. major and L. tropica.13 The species-specific real-time PCR reaction consisted of a total volume of 20 ml, containing 10 ng of extracted DNA, 0.5 mM primer 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 distilled 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
2 of 5
Confirmation of real-time PCR results by sequencing of ITS1 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.14 PCR 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’ protocol, 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. nlm.nih.gov/BLAST).
Results 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 realtime PCR assay (Table 3). The CT value of genus-specific real-time PCR assay was found to be between 20.1 and 37.1 with a median threshold of 35 cycles. On average the R2 value 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. tropica (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 cases with L. donovani, 12 (63%, 12/19) were Syrian and 7 (37% 7/ 19) Turkish. The study group did not have any Iranian, Iraqi and Afghani refugees.
Downloaded from http://trstmh.oxfordjournals.org/ at Belgorod State University on February 11, 2014
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.
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 rates were 208C/s except for the extension and the final steps, that 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 CT values were determined and the standard curves were calculated using the Rotor-Gene 6.1.93 software.
Transactions of the Royal Society of Tropical Medicine and Hygiene
Table 1. The results of genus-specific real-time PCR assay in cutaneous leishmaniasis cases Locality
Positive, %
Negative, %
Total, %
Adana Hatay S¸anlıurfa Gaziantep Kahramanmaras¸ Diyarbakır Total
28.7 (48/167) 13.2 (22/167) 10.8 (18/167) 6.0 (10/167) 3.0 (5/167) 2.4 (4/167) 64.1 (107/167)
20.4 (34/167) 5.4 (9/167) 3.6 (6/167) 3.0 (5/167) 1.8 (3/167) 1.8 (3/167) 35.9 (60/167)
49.1 (82/167) 18.5 (31/167) 14.4 (24/167) 9.0 (15/167) 4.8 (8/167) 4.2 (7/167) 100 (167/167)
Locality
Leishmania tropica, %
Leishmania major, %
Leishmania infantum, %
Leishmania donovani, %
Adana Hatay S¸anlıurfa Gaziantep Kahramanmaras¸ Diyarbakır Total
42 (20/48) 18 (4/22) 33 (6/18) 60 (6/10) 100 (5/5) 100 (4/4) 42.1 (45/107)
20 (10/48) 36 (8/22) 28 (5/18) 0 0 0 21.5 (23/107)
38 (18/48) 45 (10/22) 39 (7/18) 40 (4/10) 0 0 36.5 (39/107)
0 0 0 0 0 0 0
Table 3. The results of genus-specific real-time PCR assay in visceral leishmaniasis cases Locality
Positive, %
Negative, %
Total, %
Adana Hatay S¸anlıurfa Gaziantep Diyarbakır Kahramanmaras¸ Total
26.6 (30/113) 15.9 (18/113) 8.0 (9/113) 2.6 (3/113) 1.8 (2/113) 0.9 (1/113) 5.8 (63/113)
28.3 (32/113) 2.6 (3/113) 4.4 (5/113) 3.5 (4/113) 2.6 (3/113) 2.6 (3/113) 44.3 (50/113)
54.9 (62/113) 18.6 (21/113) 12.4 (14/113) 6.2 (7/113) 4.4 (5/113) 3.5 (4/113) 100 (113/113)
Table 4. The results of species-specific real-time PCR assay in visceral leishmaniasis cases Locality
Leishmania tropica, %
Leishmania major, %
Leishmania infantum, %
Leishmania donovani, %
Adana Hatay S¸anlıurfa Gaziantep Diyarbakır Kahramanmaras¸ Total
10 (3/30) 11 (2/18) 11 (1/9) 0 0 0 10 (6/63)
0 0 0 0 0 0 0
50 (15/30) 56 (10/18) 78 (7/9) 100 (3/3) 100 (2/2) 100 (1/1) 60 (38/63)
40 (12/30) 33 (6/18) 11 (1/9) 0 0 0 30 (19/63)
3 of 5
Downloaded from http://trstmh.oxfordjournals.org/ at Belgorod State University on February 11, 2014
Table 2. The results of species-specific real-time PCR assay in cutaneous leishmaniasis cases
I. S. Koltas et al.
The CT values 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 R2 value of the standard curve was .0.99 in all real-time PCR assays.
Results of ITS1 DNA sequencing
Discussion 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.15,16 L. tropica, L. infantum and L. major have been detected however, L. donovani has not been previously identified in Turkey.17 In our previous study carried out in 2011, we could not identify any signs of L. major in southern Turkey.9 However, in this current study, we were able to isolate L. major from CL cases in southern Turkey. 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 ecological and climatic conditions exist. Global warming has had a severe effect on most species and on Phlebotomus in particular.18,19 Secondly, 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.20,21 Many refugees have been suffering 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. tropica, which have been known to cause zoonotic VL and
4 of 5
Authors’ contributions: ISK conceived the study, analysed the data and wrote the sections on context; FE assisted in the study conception and
Downloaded from http://trstmh.oxfordjournals.org/ at Belgorod State University on February 11, 2014
Each clinical specimen and ATCC reference strain of Leishmania species (L. donovani, L. infantum, L. tropica and L. major) was identical 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 Leishmania isolates from VL revealed a 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.
anthroponotic CL in western and southeastern Turkey, respectively. 11 The 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.22 P. 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 sequencing of the Leishmania genome have increasingly suggested new approaches regarding the treatment, prognosis and control of the disease. 24 It 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 CL without VL history, while L. tropica was identified in VL without 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.10,11 Toz et al. reported 52.5% (117/223) L. infantum in CL cases.11 Our 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. tropica among 64 bone marrow/spleen samples while Toz et al. found 7% (6/92) L. tropica among 92 human and canine VL cases.10,11 They reported a rare causative agent of VL while we found 9.5% (6/63) L. tropica among 63 VL cases using speciesspecific real-time PCR and ITS1 DNA sequencing. This percentage detection is higher than those of the other studies, which suggests that L. tropica is the main causative agent of VL in southern Turkey. The main limitation of the study is related to the methodology used in that we could not perform a zymodem analysis for strains. The other limitation is that we did not investigate the relation between Leishmania species and the clinical characteristics of leishmaniasis. Using species-specific real-time PCR and ITS1 DNA sequencing, we found that L. infantum, L. major, L. tropica and L. donovani were prevalent in southern Turkey. This result indicates that Leishmania species show a cosmopolitan distribution in this region. In addition the results of the study on the diagnosis of CL and VL contribute to our current understanding of leishmaniasis epidemiology in southern Turkey. However, future studies should be carried out to further explore these issues.
Transactions of the Royal Society of Tropical Medicine and Hygiene
analysed the samples using molecular methods; DA took bone marrow 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.
11 Toz SO, Culha G, Zeyrek FY et al. A real-time ITS1-PCR based method in the diagnosis and species identification of Leishmania parasite from human and dog clinical samples in Turkey. Plos Negl Trop Dis 2013;7:e2205.
Funding: Funding was provided by the Cukurova University Research Grant [TF.2010.BAP.36].
12 Nicolas L, Prina R, Lang T et al. Real-Time PCR for detection and quantitation of Leishmania in mouse tissues. J Clin Microbiol, 2002;40:1166–69.
Competing interesting: None declared. Ethical approval: The Ethical Committee of the Medicine Faculty of Cukurova University gave approval for this study.
14 Scho¨nian G, Nasereddin A, Dinse N et al. PCR diagnosis and characterization of Leishmania in local and imported clinical samples. Diagn Microbiol Infect Dis 2003;47:349–58.
1 Minodier P, Parola P. Cutaneous leishmaniasis treatment. Travel Med Infect Dis 2007;5:150–8.
15 Khiami A, Dereure J, Pratlong F et al. Human cutaneous leishmaniasis caused by Leishmania major MON-26 in the region of Damascus (Syria). Bull Soc Pathol Exot 1991;84:340–44.
2 Stevenson LG, Fedorko DP, Zelazny AM. An enhanced method for the identification of Leishmania spp using real-time polymerase chain reaction and sequence analysis of the 7SL RNA gene region. Diagn Microbiol Infect Dis 2010;66:432–5.
16 Al-Diwany LJ, Al-Awkati NA, Atia M et al. Concomitant natural infection with L. donovani and L. major: a case report from Iraq. Soz Praventivmed 1995;40:234–38.
3 Mohammadiha A, Mohebali M, Haghighi A et al. Comparison of real-time PCR and conventional PCR with two DNA targets for detection of Leishmania (Leishmania) infantum infection in human and dog blood samples. Exp Parasitol 2013;133:89–94. 4 van der Meide W, Guerra J, Schoone G et al. Comparison between quantitative nucleic acid sequence-based amplification, real-time reverse transcriptase PCR, and real-time PCR for quantification of Leishmania parasites. J Clin Microbiol 2008;46:73–8. 5 Uzun S, Uslular C, Yu¨cel A et al. Cutaneous leishmaniasis: evaluation of 3,074 cases in the Cukurova region of Turkey. Br J Dermatol 1999;140:347–50. 6 Demirel R, Erdog˘an S. Determination of high risk regions of cutaneous leishmaniasis in Turkey using spatial analysis. Turkiye Parazitol Derg 2009;33:8–14. 7 Votypka J, Kasap OE, Volf P et al. Risk factors for cutaneous leishmaniasis in Cukurova region, Turkey. Trans R Soc Trop Med Hyg 2012;106:186–90.
17 Akman L, Aksu HS, Wang RQ et al. Multi-site DNA polymorphism analyses of Leishmania isolates define their genotypes predicting clinical epidemiology of leishmaniasis in a specific region. J Eukaryot Microbiol 2000;47:545–54. 18 Ok UZ, Balcioglu IC, Taylan Ozkan A et al. Leishmaniasis in Turkey. Acta Trop 2002;84:43–8. 19 Alexander LV, Zhang X, Peterson TC et al. Global observed changes in daily climate extremes of temperature and precipitation. JGR-Atmospheres, 2006;111:1–65. 20 http://data.unhcr.org/syrianrefugees/country.php?id=224. (accessed 29 October 2013)) 21 http://www.unhcr.org.tr/uploads/root/unhcr_-_2_million_refugees. pdf. [accessed 09 September 2013] 22 Postigo JA. Leishmaniasis in the world health organization eastern Mediterranean region. Int J Antimcrob Agents 2010;36:62–5.
8 Toz SO, Nasereddin A, Ozbel Y et al. Leishmaniasis in Turkey: molecular characterization of Leishmania from human and canine clinical samples. Trop Med Int Health 2009;14:1401–6.
23 Atakan E, Akbaba M, Su¨toluk Z et al. Population density of Phlebotomus (Diptera: Psychodidae; Phlebotomine) species and their relationship with cutaneous leishmaniasis in Hocalli and Turunc¸lu Villages (Adana). Tu¨rkiye Parazitol Derg 2010;34:106–111.
9 Eroglu F, Koltas IS, Genc A. Identification of causative species in cutaneous leishmaniasis patients using PCR-RFLP. J Bacteriol Parasitol 2011;2:3.
24 Monbrison F, Mihoubi I, Picot S. Real-time PCR assay for the identification of cutaneous Leishmania parasite species in Constantine region of Algeria. Acta Trop 2007;102:79–83.
10 Alborzi A, Rasouli M, Shamsizadeh A. Leishmania tropica-isolated patient with visceral leishmaniasis in southern Iran. Am J Trop Med Hyg 2006;74:306–7.
25 Schallig HD, Oskam L. Molecular biological applications in the diagnosis and control of leishmaniasis and parasite identification. Trop Med Int Health 2002;7:641–51.
5 of 5
Downloaded from http://trstmh.oxfordjournals.org/ at Belgorod State University on February 11, 2014
References
13 Nicolas L, Milon G, Prina E. Rapid differentiation of Old World Leishmania species by LightCycler polymerase chain reaction and melting curve analysis. J Microbiol Methods 2000;51:295–99.
ORIGINAL ARTICLE
Trans R Soc Trop Med Hyg doi:10.1093/trstmh/trt119
The emergence of Leishmania major and Leishmania donovani in southern Turkey Ismail S. Koltasa,*, Fadime Eroglua, Derya Alabazb and Soner Uzunc a
Department 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
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. major and 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 were taken from CL and VL-suspected cases, respectively. Samples were analysed through real-time PCR and ITS1 DNA sequencing. 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 confirmed with Leishmania ITS1 DNA sequencing. Conclusions: This study revealed that in southern Turkey, L. major and 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
Introduction The identification of Leishmania species is important for the determination 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 species.2 Currently, 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.3 These methods can be applied for the purpose 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,5 with CL caused by L. tropica and VL caused by L. infantum.6,7 Recent studies have reported the presence of L. infantum in CL cases without a VL history.8,9 There have also been reports of the presence of L. tropica in
VL cases without a CL history in both Iran and Turkey.10,11 This study, 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 neighbouring 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
# The Author 2014. Published by Oxford University Press on behalf of Royal Society of Tropical Medicine and Hygiene. All rights reserved. For permissions, please e-mail: [email protected].
1 of 5
Downloaded from http://trstmh.oxfordjournals.org/ at Belgorod State University on February 11, 2014
*Corresponding author: Tel: +90 535 3059393; E-mail: [email protected]
I. S. Koltas et al.
cities such as Hatay 18.5% (21/113), S¸anlıurfa 12.4% (14/113), Gaziantep 6.2% (7/113), Diyarbakır 4.5% (5/113) and 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
Genus-specific real-time PCR Assay All samples (167 CL and 113 VL) were analysed using the genusspecific 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).12 The 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, extension 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 CT values were automatically determined by the Rotor-Gene 6.1.93 software using default parameters. 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 conserved region of the Leishmania kDNA minicircle, were used to allow the amplification of L. donovani, L. infantum, L. major and L. tropica.13 The species-specific real-time PCR reaction consisted of a total volume of 20 ml, containing 10 ng of extracted DNA, 0.5 mM primer 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 distilled 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
2 of 5
Confirmation of real-time PCR results by sequencing of ITS1 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.14 PCR 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’ protocol, 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. nlm.nih.gov/BLAST).
Results 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 realtime PCR assay (Table 3). The CT value of genus-specific real-time PCR assay was found to be between 20.1 and 37.1 with a median threshold of 35 cycles. On average the R2 value 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. tropica (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 cases with L. donovani, 12 (63%, 12/19) were Syrian and 7 (37% 7/ 19) Turkish. The study group did not have any Iranian, Iraqi and Afghani refugees.
Downloaded from http://trstmh.oxfordjournals.org/ at Belgorod State University on February 11, 2014
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.
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 rates were 208C/s except for the extension and the final steps, that 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 CT values were determined and the standard curves were calculated using the Rotor-Gene 6.1.93 software.
Transactions of the Royal Society of Tropical Medicine and Hygiene
Table 1. The results of genus-specific real-time PCR assay in cutaneous leishmaniasis cases Locality
Positive, %
Negative, %
Total, %
Adana Hatay S¸anlıurfa Gaziantep Kahramanmaras¸ Diyarbakır Total
28.7 (48/167) 13.2 (22/167) 10.8 (18/167) 6.0 (10/167) 3.0 (5/167) 2.4 (4/167) 64.1 (107/167)
20.4 (34/167) 5.4 (9/167) 3.6 (6/167) 3.0 (5/167) 1.8 (3/167) 1.8 (3/167) 35.9 (60/167)
49.1 (82/167) 18.5 (31/167) 14.4 (24/167) 9.0 (15/167) 4.8 (8/167) 4.2 (7/167) 100 (167/167)
Locality
Leishmania tropica, %
Leishmania major, %
Leishmania infantum, %
Leishmania donovani, %
Adana Hatay S¸anlıurfa Gaziantep Kahramanmaras¸ Diyarbakır Total
42 (20/48) 18 (4/22) 33 (6/18) 60 (6/10) 100 (5/5) 100 (4/4) 42.1 (45/107)
20 (10/48) 36 (8/22) 28 (5/18) 0 0 0 21.5 (23/107)
38 (18/48) 45 (10/22) 39 (7/18) 40 (4/10) 0 0 36.5 (39/107)
0 0 0 0 0 0 0
Table 3. The results of genus-specific real-time PCR assay in visceral leishmaniasis cases Locality
Positive, %
Negative, %
Total, %
Adana Hatay S¸anlıurfa Gaziantep Diyarbakır Kahramanmaras¸ Total
26.6 (30/113) 15.9 (18/113) 8.0 (9/113) 2.6 (3/113) 1.8 (2/113) 0.9 (1/113) 5.8 (63/113)
28.3 (32/113) 2.6 (3/113) 4.4 (5/113) 3.5 (4/113) 2.6 (3/113) 2.6 (3/113) 44.3 (50/113)
54.9 (62/113) 18.6 (21/113) 12.4 (14/113) 6.2 (7/113) 4.4 (5/113) 3.5 (4/113) 100 (113/113)
Table 4. The results of species-specific real-time PCR assay in visceral leishmaniasis cases Locality
Leishmania tropica, %
Leishmania major, %
Leishmania infantum, %
Leishmania donovani, %
Adana Hatay S¸anlıurfa Gaziantep Diyarbakır Kahramanmaras¸ Total
10 (3/30) 11 (2/18) 11 (1/9) 0 0 0 10 (6/63)
0 0 0 0 0 0 0
50 (15/30) 56 (10/18) 78 (7/9) 100 (3/3) 100 (2/2) 100 (1/1) 60 (38/63)
40 (12/30) 33 (6/18) 11 (1/9) 0 0 0 30 (19/63)
3 of 5
Downloaded from http://trstmh.oxfordjournals.org/ at Belgorod State University on February 11, 2014
Table 2. The results of species-specific real-time PCR assay in cutaneous leishmaniasis cases
I. S. Koltas et al.
The CT values 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 R2 value of the standard curve was .0.99 in all real-time PCR assays.
Results of ITS1 DNA sequencing
Discussion 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.15,16 L. tropica, L. infantum and L. major have been detected however, L. donovani has not been previously identified in Turkey.17 In our previous study carried out in 2011, we could not identify any signs of L. major in southern Turkey.9 However, in this current study, we were able to isolate L. major from CL cases in southern Turkey. 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 ecological and climatic conditions exist. Global warming has had a severe effect on most species and on Phlebotomus in particular.18,19 Secondly, 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.20,21 Many refugees have been suffering 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. tropica, which have been known to cause zoonotic VL and
4 of 5
Authors’ contributions: ISK conceived the study, analysed the data and wrote the sections on context; FE assisted in the study conception and
Downloaded from http://trstmh.oxfordjournals.org/ at Belgorod State University on February 11, 2014
Each clinical specimen and ATCC reference strain of Leishmania species (L. donovani, L. infantum, L. tropica and L. major) was identical 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 Leishmania isolates from VL revealed a 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.
anthroponotic CL in western and southeastern Turkey, respectively. 11 The 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.22 P. 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 sequencing of the Leishmania genome have increasingly suggested new approaches regarding the treatment, prognosis and control of the disease. 24 It 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 CL without VL history, while L. tropica was identified in VL without 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.10,11 Toz et al. reported 52.5% (117/223) L. infantum in CL cases.11 Our 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. tropica among 64 bone marrow/spleen samples while Toz et al. found 7% (6/92) L. tropica among 92 human and canine VL cases.10,11 They reported a rare causative agent of VL while we found 9.5% (6/63) L. tropica among 63 VL cases using speciesspecific real-time PCR and ITS1 DNA sequencing. This percentage detection is higher than those of the other studies, which suggests that L. tropica is the main causative agent of VL in southern Turkey. The main limitation of the study is related to the methodology used in that we could not perform a zymodem analysis for strains. The other limitation is that we did not investigate the relation between Leishmania species and the clinical characteristics of leishmaniasis. Using species-specific real-time PCR and ITS1 DNA sequencing, we found that L. infantum, L. major, L. tropica and L. donovani were prevalent in southern Turkey. This result indicates that Leishmania species show a cosmopolitan distribution in this region. In addition the results of the study on the diagnosis of CL and VL contribute to our current understanding of leishmaniasis epidemiology in southern Turkey. However, future studies should be carried out to further explore these issues.
Transactions of the Royal Society of Tropical Medicine and Hygiene
analysed the samples using molecular methods; DA took bone marrow 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.
11 Toz SO, Culha G, Zeyrek FY et al. A real-time ITS1-PCR based method in the diagnosis and species identification of Leishmania parasite from human and dog clinical samples in Turkey. Plos Negl Trop Dis 2013;7:e2205.
Funding: Funding was provided by the Cukurova University Research Grant [TF.2010.BAP.36].
12 Nicolas L, Prina R, Lang T et al. Real-Time PCR for detection and quantitation of Leishmania in mouse tissues. J Clin Microbiol, 2002;40:1166–69.
Competing interesting: None declared. Ethical approval: The Ethical Committee of the Medicine Faculty of Cukurova University gave approval for this study.
14 Scho¨nian G, Nasereddin A, Dinse N et al. PCR diagnosis and characterization of Leishmania in local and imported clinical samples. Diagn Microbiol Infect Dis 2003;47:349–58.
1 Minodier P, Parola P. Cutaneous leishmaniasis treatment. Travel Med Infect Dis 2007;5:150–8.
15 Khiami A, Dereure J, Pratlong F et al. Human cutaneous leishmaniasis caused by Leishmania major MON-26 in the region of Damascus (Syria). Bull Soc Pathol Exot 1991;84:340–44.
2 Stevenson LG, Fedorko DP, Zelazny AM. An enhanced method for the identification of Leishmania spp using real-time polymerase chain reaction and sequence analysis of the 7SL RNA gene region. Diagn Microbiol Infect Dis 2010;66:432–5.
16 Al-Diwany LJ, Al-Awkati NA, Atia M et al. Concomitant natural infection with L. donovani and L. major: a case report from Iraq. Soz Praventivmed 1995;40:234–38.
3 Mohammadiha A, Mohebali M, Haghighi A et al. Comparison of real-time PCR and conventional PCR with two DNA targets for detection of Leishmania (Leishmania) infantum infection in human and dog blood samples. Exp Parasitol 2013;133:89–94. 4 van der Meide W, Guerra J, Schoone G et al. Comparison between quantitative nucleic acid sequence-based amplification, real-time reverse transcriptase PCR, and real-time PCR for quantification of Leishmania parasites. J Clin Microbiol 2008;46:73–8. 5 Uzun S, Uslular C, Yu¨cel A et al. Cutaneous leishmaniasis: evaluation of 3,074 cases in the Cukurova region of Turkey. Br J Dermatol 1999;140:347–50. 6 Demirel R, Erdog˘an S. Determination of high risk regions of cutaneous leishmaniasis in Turkey using spatial analysis. Turkiye Parazitol Derg 2009;33:8–14. 7 Votypka J, Kasap OE, Volf P et al. Risk factors for cutaneous leishmaniasis in Cukurova region, Turkey. Trans R Soc Trop Med Hyg 2012;106:186–90.
17 Akman L, Aksu HS, Wang RQ et al. Multi-site DNA polymorphism analyses of Leishmania isolates define their genotypes predicting clinical epidemiology of leishmaniasis in a specific region. J Eukaryot Microbiol 2000;47:545–54. 18 Ok UZ, Balcioglu IC, Taylan Ozkan A et al. Leishmaniasis in Turkey. Acta Trop 2002;84:43–8. 19 Alexander LV, Zhang X, Peterson TC et al. Global observed changes in daily climate extremes of temperature and precipitation. JGR-Atmospheres, 2006;111:1–65. 20 http://data.unhcr.org/syrianrefugees/country.php?id=224. (accessed 29 October 2013)) 21 http://www.unhcr.org.tr/uploads/root/unhcr_-_2_million_refugees. pdf. [accessed 09 September 2013] 22 Postigo JA. Leishmaniasis in the world health organization eastern Mediterranean region. Int J Antimcrob Agents 2010;36:62–5.
8 Toz SO, Nasereddin A, Ozbel Y et al. Leishmaniasis in Turkey: molecular characterization of Leishmania from human and canine clinical samples. Trop Med Int Health 2009;14:1401–6.
23 Atakan E, Akbaba M, Su¨toluk Z et al. Population density of Phlebotomus (Diptera: Psychodidae; Phlebotomine) species and their relationship with cutaneous leishmaniasis in Hocalli and Turunc¸lu Villages (Adana). Tu¨rkiye Parazitol Derg 2010;34:106–111.
9 Eroglu F, Koltas IS, Genc A. Identification of causative species in cutaneous leishmaniasis patients using PCR-RFLP. J Bacteriol Parasitol 2011;2:3.
24 Monbrison F, Mihoubi I, Picot S. Real-time PCR assay for the identification of cutaneous Leishmania parasite species in Constantine region of Algeria. Acta Trop 2007;102:79–83.
10 Alborzi A, Rasouli M, Shamsizadeh A. Leishmania tropica-isolated patient with visceral leishmaniasis in southern Iran. Am J Trop Med Hyg 2006;74:306–7.
25 Schallig HD, Oskam L. Molecular biological applications in the diagnosis and control of leishmaniasis and parasite identification. Trop Med Int Health 2002;7:641–51.
5 of 5
Downloaded from http://trstmh.oxfordjournals.org/ at Belgorod State University on February 11, 2014
References
13 Nicolas L, Milon G, Prina E. Rapid differentiation of Old World Leishmania species by LightCycler polymerase chain reaction and melting curve analysis. J Microbiol Methods 2000;51:295–99.
