The First Record of Zoonotic Genes of Cutaneous Leishmaniasis among Human, Dogs, and Sandflies by Nested Polymerase Chain Reaction and Phylogenetic Analyses

Rasha Alsaad; May Hameed

doi:10.3889/oamjms.2021.6639

Authors

Rasha Alsaad Department of Microbiology, University of Misan, Faculty of Medicine, Misan, Iraq; Department of Parasitology, University of Baghdad, College of Veterinary Medicine, Baghdad, Iraq https://orcid.org/0000-0002-9693-9009
May Hameed Department of Parasitology, University of Baghdad, College of Veterinary Medicine, Baghdad, Iraq

DOI:

https://doi.org/10.3889/oamjms.2021.6639

Keywords:

Cutaneous leishmaniasis, Leishmania major, Leishmania tropica, Lmj4/Uni21 primer, LITSR/L5.8S primer, Canine leishmaniasis

Abstract

Cutaneous leishmaniasis (CL) is one of the zoonotic diseases that is caused by protozoa of the genus Leishmania. The study aimed to diagnosed CL in human, dogs, and sandflies by PCR, and identification the zoonotic gene of CL by the nested PCR technique. A total of 100 patients with CL, 237 of owned-dogs, and 147 females sandflies collected. (88%) of humans samples, (95.77% skin biopsies and 20.69% of blood samples) of dogs, and (40.58%) of sandflies tissues were positive for L. major, while L. tropica infection was positive in (12%) of humans, in (4.23% symptomatic, and 6.89% asymptomatic) of dogs, and in (27.54%) of sandflies samples. The sequence ID of the local L. major in human were registered in NCBI as (MW421598.1, MW421599.1, MW421600.1), in dogs (MW421601.1, MW421602.1, MW421603.1), and sandflies (MW421604.1, MW421605.1, MW421606.1). While L. tropica in human were registered in NCBI as (MW421604.1, MW421605.1, MW421606.1), in dogs (MW421428.1, MW421429.1), and in sandflies (MW421430.1, MW421431.1). To our knowledge, this is the first study that contributes to the diagnosis of CL spp. in three different hosts (human, dogs, and sandflies) at the same time, particularly in Iraq and in Middle East countries.

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References

Centers for Disease Control and Prevention. Update CL in US. military personnel. Southwest Central Asia, 2011-2012. MMWR Morb Mortal Wkly Rep. 2012;53(12):264-5. PMid:15057192

Hepburn NC. Cutaneous leishmaniasis: An overview. J Postgrad Med. 2003;49(1):50-4. PMid:12865571 DOI: https://doi.org/10.4103/0022-3859.928

Markle WH, Makhoul K. Cutaneous Leishmaniasis Recognition and Treatment. Am Fam Physician. 2004;69(6):1455-60. PMid:15053410

Mandell GL, Bennett JE, Dolin R. In: Mandell D, Bennett JE, editors. Principles and Practice of Infectious Diseases. 6th ed. Amsterdam, Netherlands: Elsevier; 2005. p. 2428-42. https://doi.org/10.1086/655696 DOI: https://doi.org/10.1086/655696

Kumar A, Samant M. DNA vaccine against visceral leishmaniasis: A promising approach for prevention and control. Parasite Immunol. 2016;38(5):273-81. https://doi.org/10.1111/pim.12315 PMid:27009772 DOI: https://doi.org/10.1111/pim.12315

Filho JD, Brazil RP. Relationships of new world phlebotomine sand flies (Diptera: Psychodidae) based on fossil evidence. Mem Inst Oswaldo Cruz. 2003;98(Suppl 1):145-9. https://doi.org/10.1590/s0074-02762003000900022 PMid:12687775 DOI: https://doi.org/10.1590/S0074-02762003000900022

González U, Pinart M, Reveiz L, Alvar J. Interventions for old world cutaneous leishmaniasis. Cochrane Database Syst Rev. 2008;4:CD005067. https://doi.org/10.1002/14651858.cd005067.pub3 PMid:18843677 DOI: https://doi.org/10.1002/14651858.CD005067.pub3

World Health Organization. Global Leishmaniasis Surveillance, 2017-2018, and First Report on 5 Additional Indicators. Geneva: World Health Organization; 2018.

Al-Samarai AM, Al-Obaidi HS. Cutaneous leishmaniasis in Iraq. J Infect Dev Ctries. 2009;3(2):123-9. PMid:19755742 DOI: https://doi.org/10.3855/jidc.59

Habeeb MA. A Systematic, Ecological and Microbial Studies of the Sandflies (Diptera: Psychodidae; Phlebotominae) in Basrah Governorate, Iraq. Basrah: Thesis The University of Basrah; 2005. https://doi.org/10.21276/ijaq.2013.10.1.5 DOI: https://doi.org/10.21276/ijaq.2013.10.1.5

Dantas-Torres F, de Brito ME, Brandão-Filho SP. Seroepidemiological survey on canine leishmaniasis among dogs from an urban area of Brazil. Vet Parasitol. 2006;140(1-2):54-60. https://doi.org/10.1016/j.vetpar.2006.03.008 PMid:16621286 DOI: https://doi.org/10.1016/j.vetpar.2006.03.008

Elbihari S, Kawasmeh ZA, Al Naiem AH. Possible reservoir host(s) of zoonotic cutaneous leishmaniasis in Al-Hassa oasis, Saudi Arabia. Ann Trop Med Parasitol. 1984;78(5):543-5. https://doi.org/10.1080/00034983.1984.11811861 PMid:6395818 DOI: https://doi.org/10.1080/00034983.1984.11811861

Bamorovat M, Sharifi I, Aflatoonian MR, Sharifi H, Karamoozian A, Sharifi F, et al. Risk factors for anthroponotic cutaneous leishmaniasis in unresponsive and responsive patients in a major focus, Southeast of Iran. PLoS One. 2018;13(2):e0192236. https://doi.org/10.1371/journal. pone.0192236 PMid:29415078 DOI: https://doi.org/10.1371/journal.pone.0192236

Bañuls AL, Hide M, Prugnolle F. Leishmania and the leishmaniases: A parasite genetic update and advances in taxonomy, epidemiology, and pathogenicity in human. Adv Parasitol. 2007;64:1-458. https://doi.org/10.1016/s0065-308x(06)64001-3 PMid:17499100 DOI: https://doi.org/10.1016/S0065-308X(06)64001-3

Yehia L, Adib-Houreih M, Raslan WF, Kibbi AG, Loya A, Firooz A, et al. Molecular diagnosis of cutaneous leishmaniasis and species identification: Analysis of 122 biopsies with varied parasite index. J Cutan Pathol. 2012;39(3):347-55. https://doi.org/10.1111/j.1600-0560.2011.01861.x PMid:22335594 DOI: https://doi.org/10.1111/j.1600-0560.2011.01861.x

Parvizi P, Ready PD. Nested PCRs and sequencing of nuclear ITS-rDNA fragments detect three Leishmania species of gerbils in sandflies from Iranian foci of zoonotic cutaneous leishmaniasis. Trop Med Int Health. 2008;13(9):1159-71. https://doi.org/10.1111/j.1365-3156.2008.02121.x PMid:18631311 DOI: https://doi.org/10.1111/j.1365-3156.2008.02121.x

Montalvo AM, Fraga J, Monzote L, Montano I, De Doncker S, Dujardin JC, et al. Heat-shock protein 70 PCR-RFLP: A universal simple tool for Leishmania species discrimination in the new and old world. Parasitology. 2010;137(8):1159-68. https://doi.org/10.1017/s0031182010000089 PMid:20441679 DOI: https://doi.org/10.1017/S0031182010000089

Da Silva LA, de Sousa CS, da Graca GC, Porrozzi R, Cupolillo E. Sequence analysis and PCR-RFLP profiling of the hsp70 gene as a valuable tool for identifying Leishmania species associated with human leishmaniasis in Brazil. Infect Genet Evol. 2010;10(1):77-83. https://doi.org/10.1016/j.meegid.2009.11.001 PMid:19913112 DOI: https://doi.org/10.1016/j.meegid.2009.11.001

Sambrook J, Russell D. Molecular Cloning: A Laboratory Manual. 3rd ed. Cold Spring Harbor: Cold Spring Harbor Laboratory Press; 2001. https://doi.org/10.5962/bhl.title.49161 DOI: https://doi.org/10.5962/bhl.title.49161

Akhoundi M, Kuhls K, Cannet A, Votýpka J, Marty P, Delaunay P, et al. A historical overview of the classification, evolution, and dispersion of Leishmania parasites and sandflies. PLoS Negl Trop Dis. 2016;10(3):e0004349. https://doi.org/10.1371/journal. pntd.0004349 PMid:26937644 DOI: https://doi.org/10.1371/journal.pntd.0004349

Van der Auwera G, Ravel C, Verweij JJ, Bart A, Schönian G, Felger I. Evaluation of foursingle-locus markers for Leishmania species discrimination by sequencing. J Clin Microbiol. 2014;52(4):1098-104. https://doi.org/10.1128/jcm.02936-13 PMid:24452158 DOI: https://doi.org/10.1128/JCM.02936-13

Mouttaki T, Morales-Yuste M, Merino-Espinosa G, Chiheb S, Fellah H, Martin-Sanchez J, et al. Molecular diagnosis of cutaneous leishmaniasis and identification of the causative Leishmania species in Morocco by using three PCR-based assays. Parasites Vectors. 2014;7:420. https://doi.org/10.1186/1756-3305-7-420 DOI: https://doi.org/10.1186/1756-3305-7-420

Reithinger R, Dujardin JC. Molecular diagnosis of leishmaniasis: Current status and future applications. J Clin Microbiol 2007;45(1):21-5. https://doi.org/10.1128/jcm.02029-06 PMid:17093038 DOI: https://doi.org/10.1128/JCM.02029-06

El-Beshbishy HA, Al-Ali KH, El-Badry AA. Molecular characterization of cutaneous leishmaniasis in Al-Madinah Al-Munawarah Province, Western Saudi Arabia. Int J Infect Dis. 2013;17(5):e334-8. https://doi.org/10.1016/j.ijid.2012.11.015 PMid:23253909 DOI: https://doi.org/10.1016/j.ijid.2012.11.015

Azizi K, Soltani A, Alipour H. Molecular detection of Leishmania isolated from cutaneous leishmaniasis patients in Jask County, Hormozgan Province, Southern Iran, 2008. Asian Pac J Trop Med 2012;5(7):514-7. https://doi.org/10.1016/s1995-7645(12)60090-x PMid:22647811 DOI: https://doi.org/10.1016/S1995-7645(12)60090-X

Khademvatan S, Saki J, Maraghi S. Comparison of traditional methods and PCR for diagnosis of cutaneous leishmaniasis in South-West of Iran. Zahedan J Res Med Sci. 2012;14(8):1-5. PMid:22347259

Azmi K, Nasereddin A, Ereqat S, Schnur L, Schonian G, Abdeen Z. Methods incorporating a polymerase chain reaction and restriction fragment length polymorphism and their use as a gold standard in diagnosing Old World cutaneous leishmaniasis. Diagn Microbiol Infect Dis. 2011;71(2):151-5. https://doi.org/10.1016/j.diagmicrobio.2011.06.004 PMid:21840670 DOI: https://doi.org/10.1016/j.diagmicrobio.2011.06.004

Kumar R, Bumb RA, Ansari NA, Mehta RD, Salotra P. Cutaneous leishmaniasis caused by Leishmania tropica in Bikaner, India: Parasite identification and characterization using molecular and immunologic tools. Am J Trop Med Hyg 2007;76(5):896-901. https://doi.org/10.4269/ajtmh.2007.76.896 PMid:17488912 DOI: https://doi.org/10.4269/ajtmh.2007.76.896

Bensoussan E, Nasereddin A, Jonas F, Schnur LF, Jaffe CL. Comparison of PCR assays for diagnosis of cutaneous leishmaniasis. J Clin Microbiol. 2006;44(4):1435-9. https://doi.org/10.1128/jcm.44.4.1435-1439.2006 PMid:16597873 DOI: https://doi.org/10.1128/JCM.44.4.1435-1439.2006

Mauricio I, Stothard A, Miles M. Leishmania donovani complex: Genotyping with the ribosomal internal transcribed spacer and the mini-exon. Parasitology. 2004;128(3):263-7. https://doi.org/10.1017/s0031182003004578 PMid:15074875 DOI: https://doi.org/10.1017/S0031182003004578

Aldifaei RS. Prevalence of Cutaneous Leishmaniasis in AL-Qadissia Province and the Evaluation of Treatment Response by Pentostam with RT-PCR, Thesis; 2013.

Alanazi AD, Puschendorf R, Alyousif MS, Al-Khalifa MS, Alharbi SA, Al-Shehri ZS, et al. Molecular detection of Leishmania spp. in skin and blood of stray dogs from endemic areas of cutaneous leishmaniasis in Saudi Arabia. Iran J Parasitol. 2019;14(2):231-9. https://doi.org/10.18502/ijpa.v14i2.1135 PMid:31543911 DOI: https://doi.org/10.18502/ijpa.v14i2.1135

Ajouad M, Es-Sette N, Hamdi S, El-Idrissi A, Riyad M, Lemrani M. Detection and molecular typing of Leishmania tropica from Phlebotomus sergenti and lesions of cutaneous leishmaniasis in an emerging focus of Morocco. Parasit Vectors. 2013;6(1):217. https://doi.org/10.1186/1756-3305-6-217 PMid:23890256 DOI: https://doi.org/10.1186/1756-3305-6-217

El Tai NO, El Fari M, Mauricio I, Miles MA, Oskam L, El Safi SH, et al. Leishmania donovani: Intraspecific polymorphisms of Sudanese isolates revealed by PCR-based analyses and DNA sequencing. Exp Parasitol. 2001;97(1):35-44. https://doi.org/10.1006/expr.2001.4592 PMid:11207112 DOI: https://doi.org/10.1006/expr.2001.4592

El Tai NO, Osman OF, El Fari M, Presber W, Schönian G. Genetic heterogeneity of ribosomal internal transcribed spacer in clinical samples of Leishmania donovani spotted on filter paper as revealed by single-strand conformation polymorphisms and sequencing. Trans R Soc Trop Med Hyg. 2000;94(5):575-9. https://doi.org/10.1016/s0035-9203(00)90093-2 PMid:11132393 DOI: https://doi.org/10.1016/S0035-9203(00)90093-2

Spanakos G, Piperaki ET, Menounos PG, Tegos N, Flemetakis A, Vakalis NC. Detection and species identification of old world Leishmania in clinical samples using a PCR based method. Trans R Soc Trop Med Hyg. 2008;102(1):46-53. https://doi.org/10.1016/j.trstmh.2007.05.019 PMid:17669452 DOI: https://doi.org/10.1016/j.trstmh.2007.05.019

Schallig HD, Oskam L. Molecular biological applications in the diagnosis and control of leishmaniasis and parasite identification. Trop Med Int Health. 2002;7(8):641. https://doi.org/10.1046/j.1365-3156.2002.00911.x PMid:12167091 DOI: https://doi.org/10.1046/j.1365-3156.2002.00911.x

Parvizi P, Moradi G, Akbari G, Farahmand M, Ready PD, Piazak N, et al. PCR detection and sequencing of parasite ITS-rDNA gene from reservoir host of zoonotic cutaneous leishmaniasis in central Iran. Parasitol Res. 2008;136(6):1289- 95. https://doi.org/10.1007/s00436-008-1220-0 PMid:18791741 DOI: https://doi.org/10.1007/s00436-008-1124-z

Es-Sette N, Ajaoud M, Bichaud L, Hamdi S, Mellouki F, Charrel RN, et al. Phlebotomus sergenti a common vector of Leishmania tropica and Toscana virus in Morocco. J Vector Borne Dis. 2014;51(2):86-90. https://doi.org/10.1186/s13071-015-0826-1 PMid:24947214 DOI: https://doi.org/10.1186/s13071-015-0826-1

Al-Huchaimi S, Al-Nafakh R, Al-Khafaji Z, Amad N, Mahmood T, Bedri S, et al. Phylogenetic analysis of sandflies populations using cytochrome b (mtCytb) gene and identification of Leishmania DNA within infected sandflies, from the city of Najaf, Iraq. J Contemp Med Sci. 2018;4(3):163-9.

Azizi K, Badzohreh A, Sarkari B, Fakoorziba MR, Kalantari M, Moemenbellah-Fard MD, et al. Nested polymerase chain reaction and sequence-based detection of Leishmania infection of sand flies in recently emerged endemic focus of zoonotic cutaneous leishmaniasis, Southern Iran. Iran J Med Sci. 2013;38(Suppl 2):156-62. https://doi.org/10.1089/vbz.2011.0872 PMid:24031105 DOI: https://doi.org/10.1089/vbz.2011.0872

Azizi K, Rassi Y, Moemenbellah-Fard MD. PCR-based detection of Leishmania major kDNA within naturally infected Phlebotomus papatasi in Southern Iran. Trans R Soc Trop Med Hyg. 2010;104(6):440-2. https://doi.org/10.1016/j.trstmh.2010.01.008 PMid:20129635 DOI: https://doi.org/10.1016/j.trstmh.2010.01.008

Davami MH, Motazedian MH, Kalantari M, Asgari Q, Badzohre A, Mohammadpour I. First microscopical and molecular-based characterization of Leishmania major within naturally infected Phlebotomus salehi ( Diptera; Psychodidae) in Fars Province, Southern Iran. Ann Trop Med Parasitol. 2011;105(7):485-91. https://doi.org/10.1016/s1201-9712(11)60330-5 PMid:22185942 DOI: https://doi.org/10.1016/S1201-9712(11)60330-5

Seray O, Toz G, Fadile Y, Hatice E, Ziya A, Aslı V, 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(5):e2205. https://doi.org/10.1371/journal.pntd.0002205 PMid:23675543 DOI: https://doi.org/10.1371/journal.pntd.0002205

Ramezany M, Sharifi I, Babaei Z, Almani PG, Heshmatkhah A, Keyhani A, et al. Geographical distribution and molecular characterization for cutaneous leishmaniasis species by sequencing and phylogenetic analyses of kDNA and ITS1 loci markers in South-Eastern Iran. Pathog Glob Health. 2018;112(3):132-41. https://doi.org/10.1080/20477724.2018.1447836 PMid:29536818 DOI: https://doi.org/10.1080/20477724.2018.1447836

Sharifi F, Sharifi I, Zarean M, Parizi MH, Aflatoonian M, Harandi MF, et al. Spatial distribution and molecular identification of Leishmania species from endemic foci of south-eastern Iran. Iran J Parasitol. 2012;7(1):45-52. PMid:23133471

Mahdy MA, Al-Mekhlafi HM, Al-Mekhlafi AM, Lim YA, Bin Shuaib NO, Azazy AA, et al. Molecular characterization of Leishmania species isolated from cutaneous leishmaniasis in Yemen. PLoS One. 2010;5(9):e12879. https://doi.org/10.1371/journal.pone.0012879 PMid:20862227 DOI: https://doi.org/10.1371/journal.pone.0012879

Mohebali M, Malmasi A, Hajjaran H, Jamshidi S, Akhoundi B, Rezaei M, et al. Disseminated leishmaniasis caused by Leishmania tropica in a puppy from Karaj, Central Iran. Iran J Parasitol. 2011;6(2):69-73. PMid:22347291

Solano-Gallego L, Miró G, Koutinas A, Cardoso L, Pennisi MG, Ferrer L, et al. LeishVet guidelines for the practical management of canine leishmaniosis. Parasit Vectors. 2011;4:86. https://doi.org/10.1186/1756-3305-4-86 PMid:21599936 DOI: https://doi.org/10.1186/1756-3305-4-86

Bamorovat M, Sharifi I, Dabiri S, Mohammadi MA, Harandi MF, Mohebali M, et al. Leishmania tropica in Stray Dogs in Southeast Iran. Iran J Public Health. 2015;44(10):1359-66. PMid:26576349

Abdulwahab AR. Genotype of Cutaneous Leishmaniasis in Iraq in Correlation with Dental Broach Smear and Histopathological Section. Baghdad: Thesis College of Medicine, University of Baghdad; 2013.

Saki J, Meamar A, Oormazdi H, Akhlaghi L, Maraghi S, Mohebali M, et al. Mini-exon genotyping of Leishmania species in Khuzestan Province, Southwest Iran. Iran J Parasitol. 2010;5(1):25-34. https://doi.org/10.5812/jjm.4805 PMid:22347232 DOI: https://doi.org/10.5812/jjm.4805

Van der Auwera G, Dujardin JC. Species typing in dermal leishmaniasis. Clin Microbiol Rev. 2015;28(2):265-94. https://doi.org/10.1128/cmr.00104-14 PMid:25672782 DOI: https://doi.org/10.1128/CMR.00104-14

Bamorovat M, Sharifi I, Mohammadi MA, Eybpoosh S, Nasibi S, Aflatoonian MR, et al. Leishmania tropica isolates from non-healed and healed patients in Iran: A molecular typing and phylogenetic analysis. Microb Pathog. 2018;116:124-9. https://doi.org/10.1016/j.micpath.2018.01.021 PMid:29353004 DOI: https://doi.org/10.1016/j.micpath.2018.01.021

Schonian G, Schnur L, El Fari M, Oskam L, Kolesnikov AA, Sokolowska-Köhler W, et al. Genetic heterogeneity in the species Leishmania tropica revealed by different PCR-based methods. Trans R Soc Trop Med Hyg. 2001;95(2):217-24. https://doi.org/10.1016/s0035-9203(01)90173-7 DOI: https://doi.org/10.1016/S0035-9203(01)90173-7

Ekşi F, Özgöztaşı O, Karslıgil T, Sağlam M. Genotyping Leishmania promastigotes isolated from patients with cutaneous leishmaniasis in South-Eastern Turkey. J Int Med Res 2017;45(1):114-22. https://doi.org/10.1177/0300060516677155. DOI: https://doi.org/10.1177/0300060516677155

Salloum T, Khalifeh I, Tokajian S. Detection, molecular typing and phylogenetic analysis of Leishmania isolated from cases of leishmaniasis among Syrian refugees in Lebanon. Parasite Epidemiol Control. 2016;1(2):159-68. https://doi.org/10.1016/j.parepi.2016.02.002 DOI: https://doi.org/10.1016/j.parepi.2016.02.002

Parvizi P, Baghban N, Novin EA, Absavaran A. Detection, identification and molecular typing of Leishmania major in Phlebotomus papatasi from a focus of zoonotic cutaneous leishmaniasis in central of Iran. Exp Parasitol. 2010;124(2):232-7. https://doi.org/10.1016/j.exppara.2009.10.004 PMid:19854172 DOI: https://doi.org/10.1016/j.exppara.2009.10.004