J Arthropod-Borne Dis, March 2018, 12(1): 67–75 L Masoori et al.: Molecular-Based Detection of … 67 http://jad.tums.ac.ir Published Online: March 18, 2018 Original Article Molecular-Based Detection of Leishmania infantum in Human Blood Samples in a New Focus of Visceral Leishmaniasis in Lorestan Province, Iran Leila Masoori 1, Farnaz Kheirandish 2, Ali Haghighi 1, Mehdi Mohebali 3,4, Behnaz Ak- houndi 3, Niloofar Taghipour 1, Latif Gachkar 5, Ali Chegeni-Sharafi 6, *Vahideh Moin- Vaziri 1 1Department of Parasitology and Mycology, School of Medicine, Shahid Beheshti University of Medical Sciences, Tehran, Iran 2Department of Parasitology and Mycology, School of Medicine, Lorestan University of Medical Sciences, Khorramabad, Iran 3Department of Medical Parasitology and Mycology, School of Public Health, Tehran University of Medical Sciences, Tehran, Iran 4Center for Research of Endemic Parasites of Iran (CREPI), Tehran University of Medical Sciences, Tehran, Iran 5Infectious Diseases and Tropical Medicine Research Center, Shahid Beheshti University of Medical Sciences, Tehran, Iran 6Department of Communication Disease Control and Prevention, Deputy of Health, Lorestan University of Medical Sciences, Lorestan, Khorramabad, Iran (Received 15 Mar 2015; accepted 19 Feb 2018) Abstract Background: The fatal form of leishmaniasis is visceral form (VL), found in some of the countries in the world. Visceral leishmaniasis has been reported sporadically from all provinces in Iran, including Lorestan. This study aimed to characterize parasite species in DAT positive and some of the DAT negative human blood samples of Delfan district, Lorestan Province, central Iran. Methods: Blood samples were collected from different geographical areas of Delfan. Serum was used for DAT test and remained part of molecular study. DNA was extracted by using DNG-plus extracted kit (Cinagen, Iran). Poly- merase chain reaction amplification of Leishmania kDNA and PCR-RFLP of ITS1 was done to identify Leishmania species. Some amplicons were sequenced, submitted to GenBank and analyzed by BLASTn. Results: Expected band of kDNA for L. infantum (720bp) was amplified in 16 out of 186 (8.6%) samples which showed previously anti-Leishmania antibody at different titers or were negative serologically. Using BLASTn, 93% similarity with L. infantum has been shown. The rDNA-ITS1 was amplified only in 9 samples (4.7%). RFLP pattern was similar to what expected for L. infantum. Conclusion: A new emerging hypo-endemic focus, caused by L. infantum, is going to be established in Delphan District, Lorestan Province. Further studies on vector and reservoirs are necessary for the region and other parts of Lorestan Province. Keywords: Visceral leishmaniasis, Leishmania infantum, kDNA, ITS1, Iran Introduction Leishmaniasis is a group of protozoan dis- eases transmitted to humans and other mam- mals by Phlebotominae sandflies and can mani- fest in different clinical forms, depending upon the infecting species of Leishmania. The dis- ease could emerge as cutaneous, mucocutane- ous or Visceral Leishmaniasis (VL) (1). Medi- terranean form of VL is defined as a zoonotic disease, VL is a huge burden on human health and society, it is caused by L. donovani complex in Asia and Africa and by L. chagasi, a synony- mous of L. infantum, in Latin America (2-4). *Corresponding author: Dr Vahideh Moin-Vaziri, E-mail: vmvaziri@gmail.com http://jad.tums.ac.ir/ J Arthropod-Borne Dis, March 2018, 12(1): 67–75 L Masoori et al.: Molecular-Based Detection of … 68 http://jad.tums.ac.ir Published Online: March 18, 2018 The leishmaniasis is found to be prevalent in some of the poorest countries in the world, and they gain lesser attention than another in- fectious disease like malaria, tuberculosis, and AIDS. They are categorized as a neglected trop- ical disease (NTD) because although not rec- ognized and prioritized, they cause significant health problems to the very poorest in socie- ty. There is little effort on the part of the glob- al community and pharmaceutical industry to invest in research and development of better and innovative therapeutic because of lack of sufficient incentives (1, 5). Leishmaniasis rank as the leading NTD in terms of mortality, with an estimated 20000– 40000 death (6, 7) and 3.3 million disability- adjusted life years (8). The most life-threating form of disease is VL, in which the pathogen disseminates to endothelial systems, like liver, spleen and bone marrow. Clinical signs and symptoms of VL generally include prolonged and irregular fe- ver associated with chills, hepatosplenomeg- aly, lymphadenopathy, progressive anemia, weight loss. More than 90% of VL cases are reported from Bangladesh, Brazil, Ethiopia, India, Sudan and South Sudan (8). Two different forms of leishmaniasis com- prising VL and CL exist in Iran. Cases of VL have been reported from all parts of the country (9). Unlike CL, which accounts for almost 20000 new cases per year (9), VL has been reported sporadically, with endemic foci lo- cated in northwestern and southern Iran, with about 100–300 new cases annually (9, 10). The diagnosis of VL is a challenging issue and several studies work to resolve problem (4). Direct agglutination test (DAT) has been applied vastly for serological studies of VL in human and animal reservoir hosts in the world as well in Iran particularly in the en- demic areas (9, 11-13). DAT is a simple, cost- effective and field applicable test and has been recommended for seroepidemiological studies as well as early and accurate diagno- sis of VL, especially in endemic areas (9, 14). As mentioned VL is reported sporadical- ly in all provinces in Iran, there are several reports of sporadic VL cases in Lorestan Prov- ince, but real status of disease is not clear in this area. Lorestan is a province of western Iran in the Zagros Mountains, the climate is gen- erally sub-humid continental with cold win- ter. A seroepidemiological study using DAT was performed on 800 collected sera from healthy population of Delphan County locat- ed in mountainous part of Lorestan Province by the same authors. Anti-Leishmania anti- body at different titers in 38 cases (4.75%) which indicate that a new focus of VL with low endemicity is going to be formed in Delphan district (15). Therefore, further studies seem necessary on Leishmania species identification, vector incrimination, as well as reservoirs. Identification of Leishmania parasites is essential for precise prognosis of the disease as well as making proper decision regarding treatment (16, 17). DNA-based methods have proven to be effective in detecting the genome of Leishmania species in different biological samples with aim of species identification (4). Among different genetic markers used for Leishmania identification, kDNA, and ITS1 vastly used in detecting parasite in different bi- ological samples (18-20). The kinetoplast DNA contains around 10000 mini-circles per cell, each around 800bp in length with an approxi- mately 600bp variable and 200bp conserved region. The heterogeneity of the variable region has been exploited to discriminate between Leishmania spp (21). ITS1-RFLP-PCR also is common in characterization of different Leish- mania spp (16, 20, 22, 23). The current study aimed to characterize Leishmania species in the peripheral blood samples which showed anti-Leishmania an- tibodies at titer ≥1:3200 (considered positive) and titers between 1:800 and 1:1600 (consid- ered suspected) and 20% of titers under 1:800 (considered negative) obtained from previous study (15), targeting kDNA and ITS1 gene. http://jad.tums.ac.ir/ J Arthropod-Borne Dis, March 2018, 12(1): 67–75 L Masoori et al.: Molecular-Based Detection of … 69 http://jad.tums.ac.ir Published Online: March 18, 2018 Materials and Methods Study area Delfan district is located in mountainous area of northwest of Lorestan Province. The altitude of study area is about 2000m above sea level and very cold in winter. The total pop- ulation was 85000 in 2009, including 1200 no- madic family. Sample preparation Totally 800 samples were collected from children ≤ 12yr old and 10% of adults by a multi-stage randomized cluster sampling in 2012. After filling out a questionnaire, 3ml of peripheral blood sample has been taken from each person. Sera of about 1ml of blood sam- ple had been used for serology study, using DAT at the School of Public Health, Tehran University of Medical Sciences, Tehran, Iran (10) and results published previously (15). Two remained ml were kept at -20 °C in the EDTA- containing tubes in Department of Parasitolo- gy, Shahid Beheshti University of Medical Sci- ences, Tehran, Iran for DNA extraction. Based on serology results (15), just 186 samples out of 800 which showed anti-Leish- mania antibody at different titers were sub- jected to molecular works, comprising 5 cases (0.62%) at titers ≥ 1:3200 considered as pos- itive, 21 samples (2.62%) at titer 1:800 to 1:1600 considered suspected and 160 samples (20%) serologically negative (out of them 146 showed no titer and 14 at titer 1:400) (Table 1). DNA Extraction Five hundred microliters of whole blood were used for DNA extraction. To remove in- terfering hemoglobin molecules from the samples prior to DNA extraction, blood was washed with PBS, even 10 times. DNA was extracted using DNG-plus extracted kit (Cinagen, Iran) based on manufacturer in- structions, DNA concentration was determined by NanoDrop (Bio wave II) at 220 and 280nm. Standard strains L. infantum: MCAN/IR/07/ Moheb.gh, L. tropica: MHOM/IR/02/Mash10 and L. major: MRHO/IR/75/ER was achieved from Department of Medical Parasitology, School of Public Health, Tehran University of Medical Sciences, Iran to monitor the reactions. PCR amplification of Leishmania kDNA For initial parasite detection, a nested- PCR was performed by using specific primers CSB2XF: (CGAGTAGCAGAAACTCCCG TTCA), CSB1XR: (ATTTTTCGCGATTTT CGCAGAACG) (external) and 13Z: (ACT GGGGGTTGGTGTAAAATAG), LiR: (TC GCAGAACGCCCCT) (internal) (21). These primers amplified a fragment of 560 to 750 bp species dependent. The amplification condi- tions of both two rounds were 94 °C for 5 min, followed by 30 cycles of denaturation at 94 °C for 60 sec, annealing at 55 °C for 60 sec and extension at 72 °C for 90sec, with a final extension step at 72 °C for 5min. Five µl of the PCR production of second rounds were visualized on a UV transilluminator fol- lowing 1.5% agarose gel electrophoresis con- taining ethidium bromide. PCR amplification for Leishmania ITS1 LITSR and L5.8 primers were used to am- plify the small subunit 5.8s rRNA of parasite genome. Specific primers, LITSR (forward: 5′-CTGGATCATTTTCCGATG-3′) and L5.8S: (reverse: 5′-TGATACCACTTATCGCACTT- 3′), then PCR products were digested by HaeIII (BsuRI) restriction enzyme following proto- col (24). After using the restriction enzyme, pattern fragment of 200, 80 and 60bp for L. infantum would be produced and visualized by 2% agarose gel electrophoresis, stained with ethidium bromide. All the procedures were monitored by standard Leishmania parasite (L. major Acc. No: JN860745, L. tropica Acc. No: EF653267 and L. infantum (Acc. No: FJ555210). http://jad.tums.ac.ir/ J Arthropod-Borne Dis, March 2018, 12(1): 67–75 L Masoori et al.: Molecular-Based Detection of … 70 http://jad.tums.ac.ir Published Online: March 18, 2018 Sequencing Some samples were sequenced, submitted to GenBank and their homology with the avail- able sequence data in GenBank was checked by using BLASTn (25). Ethics Ethics clearance was obtained from Re- search Ethical Community of Shahid Beheshti University of Medical Sciences (Approval Number: SMBU.REC.1392.302(. Results Leishmania parasite identification by kDNA- Nested-PCR In 16 samples out of 186 ones (8.6%), am- plification of kDNA was yielded by visualiz- ing band of 720bp, for L. infantum (Fig. 1). The kDNA amplification was obtained from 6 serologically negative samples, including one sample with no titer and 5 others at titer 1:400 (Table 1). The PCR products of 6 samples were se- quenced and deposited in GenBank (Acces- sion no: KJ417490, KJ417491, KJ417492, KJ417493, KJ417494, KJ417495). BLAST analysis showed 83–93% homology to L. do- novani (Accession no: AJ100780.1) and L. infantum (Accession no.NZ32847.1). Leishmania parasite identification with ITS1-PCR-RFLP Totally 9 samples (including sero-negative cases) out of 189 (4.7%) have shown Leish- mania infection by visualizing a band approx- imately 350bp in 1.5% agarose gel. Digestion of amplicons with HaeIII (BsuRI) enzyme pro- duced the fragments of 200, 80 and 60bp char- acterized as L. infantum in all samples. The re- sults also were monitored by digestion pattern in reference strain of L. infantum (Fig. 2). One PCR product was sequenced and sub- mitted to GenBank, which is accessible by Ac- cession no. KJ417496. BLAST analysis showed 94% similarity to Iranian L. infantum. (Ac- cession no: KC34730 and HQ535858.1). Fig. 1. Gel electrophoresis of kDNA-Nested-PCR products obtained from L. infantum isolates of blood samples collected from Delfan district, Lorestan Province Lane 1–3: Leishmania tropica MHOM/IR/02/ Mash10 (750bp), L. infantum MCAN/IR/07/ Moheb.gh (720bp), L. major MHOM/IR/75/ER (560bp), standard strains respectively Lane 4: Sample of current study (L. infantum) Lane 5: Molecular weight marker (100bp ladder Fermentas) Fig. 2. Digestion of ITS1-rDNA amplicons using HaeIII (BsuRI) enzyme of Leishmania isolated from blood samples collected from Delfan district, Lorestan Province Lane 1 and 7: M: Molecular weight marker (100 bp ladder Fermentas) Lane 2 and 3: Current study samples Lane 4–6: Leishmania tropica MHOM/IR/02/ Mash10, L. infantum MCAN/IR/07/Moheb.gh, L. major MHOM/IR/75/ER standard strains re- spectively http://jad.tums.ac.ir/ J Arthropod-Borne Dis, March 2018, 12(1): 67–75 L Masoori et al.: Molecular-Based Detection of … 71 http://jad.tums.ac.ir Published Online: March 18, 2018 Table 1. Number of molecular positive samples at different anti-Leishmania antibody titer among collected human blood samples from Delfan District, Lorstan Province, Iran, 2012 Antibody Titer Number of samples Molecular positive cases Percent Negative No titer 146 1 0.68 1:400 14 5 35.7 Suspected: (1:800 to 1:1600) 21 9 42.8 Positive: (≥ 1: 3200) 5 1 20 Total 186 16 8.6 Discussion VL is a life threating disease with an esti- mation of 50000 deaths in 2010 in the world (6). VL occurs sporadically in all geographical zones of Iran but it is endemic in some areas mainly located in northwestern and southern part of the country. Disease status in Lorestan Province is not clearly distinct. Our previous study in Delfan District (located in northwest of Lorestan Province) showed 3.25% sero- positive samples among 800 collected ones using DAT (15). Leishmania species identi- fication is of great importance from clinical and epidemiological point of view. As a re- sult of morphological similarities, Leishmania species could not be firmly identified using conventional methods. Main objective of cur- rent study was to characterize the Leishmania species among the samples which collected from Delfan and as a result of our previous study showed anti-Leishmania antibodies at different titers (15), targeting two different molecular markers. kDNA is introduced as a quite useful mark- er for initial screening of parasite by different researchers (26, 27). In the present study, kDNA could be a reliable marker because peripheral blood samples were used. A fragment about 720bp of kDNA was visualized in 16 out of 186 samples (8.6%), which based on the size and sequencing results they were characterized as L. infantum. This marker is used commonly in distinguishing parasite in human samples, reservoirs, and vectors, most especially when there was not enough amount of parasite in the samples (21, 23, 28, 29). Using this marker, L. infantum was isolated from sandflies in Greece by producing a fragment of 720bp of parasite genome (30). Leishmania infantum infection was identified in asymptomatic dogs in a new endemic focus of VL in Iran (31). Another useful marker which vastly used for Leishmania species identification is ITS1 (24, 32-37). In the current study, expected band of 350bp length was amplified just in 9 out of 186 samples (4.8%). Compared to kDNA positive cases (8.4%) it could be con- cluded that kDNA is quite appropriate for in- itial screening especially in case of inadequate amount of parasite in clinical samples. This marker could detect 0.01 to 0.001 parasites per ml whereas ITS1 has ability of detection of 1–6 parasites per ml (37, 38). Enzymatic digestion pattern of ITS1 amplicons was the same as what expected for L. infantum which verified the results obtained using kDNA. Both genetic markers could detect parasite in one sample considered serologically nega- tive. It was a recent infection which empha- sizes on the ability of molecular tools in early detection of parasite, especially reasoning the ability of kDNA marker in initial screening of the samples. If we had chance to follow the case, it would have shown rising titer, unfor- tunately, it was not possible for authors in spite of several attempts. DNA of Leishmania parasite also was detected in 5 samples out of 14 (35.7%) which showed anti-Leishmania antibodies at titer 1/400, which according to the interpretation of DAT results, they were considered negative. In these cases also rising http://jad.tums.ac.ir/ J Arthropod-Borne Dis, March 2018, 12(1): 67–75 L Masoori et al.: Molecular-Based Detection of … 72 http://jad.tums.ac.ir Published Online: March 18, 2018 titer was probable, if follow up was possible. Conclusion Altogether, seroepidemiology and molec- ular results of current survey showed that a new emerging hypo-endemic focus is going to be established in Delfan District, Lorestan Prov- ince, due to L. infantum. Considering the life- style, climate and massive migration for seek- ing job, necessity of further work on other ep- idemiological aspects of disease are evident, as well health education should be considered by health authorities. Acknowledgements This study was supported financially by Infectious Diseases and Tropical Medicine Center, Shahid Beheshti University of Medical Sciences, Tehran, Iran. (Project no: 04221482). This article has been extracted from the the- sis written by Ms Leila Masoori (Registration No.: 150) The authors are grateful for the sin- cere cooperation of staff of Delfan Health center (Mr Mansoori, Mr Geraeian, and Mrs Sajadi), director and staff of laboratory of Ibn- e-Sina hospital in Noor-Abad, director and staff of laboratory of Shahid-Madani Hospi- tal (especially Mr Sabzevari). The authors de- clare that there is no conflict of interests. References 1. Salam N, Al-Shaqha WM, Azzi A (2014) Leishmaniasis in the Middle East: in- cidence and epidemiology. PLoS NTD. 8(10): e3208. 2. Desjeux P (2001) The increase in risk fac- tors for leishmaniasis worldwide. Trans R Soc Trop Med Hyg. 95(3): 239–243. 3. Desjeux P (2004) Leishmaniasis. Nat Rev Microbiol. 2(9): 692. 4. Pereira MR, Rocha-Silva F, Graciele-Melo C, Lafuente CR, Magalhães T, Ca- ligiorne RB (2014) Comparison be- tween conventional and real-time PCR assays for diagnosis of visceral leish- maniasis. 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