Volume 95, Issue 5
  • ISSN: 0002-9637
  • E-ISSN: 1476-1645



This study primarily aimed to identify the causative species of cutaneous leishmaniasis (CL) in the Khyber Pakhtunkhwa Province of Pakistan and to distinguish any species-specific variation in clinical manifestation of CL. Diagnostic performance of different techniques for identifying CL was assessed. Isolates of spp. were detected by in vitro culture, polymerase chain reaction (PCR) on DNA extracted from dried filter papers and microscopic examination of direct lesion smears from patients visiting three major primary care hospitals in Peshawar. A total of 125 CL patients were evaluated. Many acquired the disease from Peshawar and the neighboring tribal area of Khyber Agency. Military personnel acquired CL while deployed in north and south Waziristan. was identified as the predominant infecting organism in this study (89.2%) followed by (6.8%) and, unexpectedly, (4.1%). These were the first reported cases of CL caused by in Pakistan. PCR diagnosis targeting kinetoplast DNA was the most sensitive diagnostic method, identifying 86.5% of all samples found positive by any other method. Other methods were as follows: ribosomal DNA PCR (78.4%), internal transcribed spacer 2 region PCR (70.3%), culture (67.1%), and microscopy (60.5%). Clinical examination reported 14 atypical forms of CL. Atypical lesions were not significantly associated with the infecting species, nor with “dry” or “wet” appearance of lesions. Findings from this study provide a platform for species typing of CL patients in Pakistan, utilizing a combination of in vitro culture and molecular diagnostics. Moreover, the clinical diversity described herein can benefit clinicians in devising differential diagnosis of the disease.

[open-access] This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.


Article metrics loading...

The graphs shown below represent data from March 2017
Loading full text...

Full text loading...



  1. Hotez PJ, Molyneux DH, Fenwick A, Ottesen E, Ehrlich Sachs S, Sachs JD, , 2006. Incorporating a rapid-impact package for neglected tropical diseases with programs for HIV/AIDS, tuberculosis, and malaria. PLoS Med 3: e102.[Crossref] [Google Scholar]
  2. Hotez PJ, Remme JH, Buss P, Alleyne G, Morel C, Breman JG, , 2004. Combating tropical infectious diseases: report of the Disease Control Priorities in Developing Countries Project. Clin Infect Dis 38: 871878.[Crossref] [Google Scholar]
  3. Desjeux P, , 2004. Leishmaniasis: current situation and new perspectives. Comp Immunol Microbiol Infect Dis 27: 305318.[Crossref] [Google Scholar]
  4. WHO, 2010. Control of the Leishmaniases: Report of a Meeting of the WHO Expert Committee on the Control of Leishmaniases. Geneva, Switzerland: World Health Organization. [Google Scholar]
  5. Alvar J, Velez ID, Bern C, Herrero M, Desjeux P, Cano J, Jannin J, den Boer M, , 2012. Leishmaniasis worldwide and global estimates of its incidence. PLoS One 7: e35671.[Crossref] [Google Scholar]
  6. Noyes HA, Reyburn H, Bailey JW, Smith D, , 1998. A nested-PCR-based schizodeme method for identifying Leishmania kinetoplast minicircle classes directly from clinical samples and its application to the study of the epidemiology of Leishmania tropica in Pakistan. J Clin Microbiol 36: 28772881. [Google Scholar]
  7. Rab MA, al Rustamani L, Bhutta RA, Mahmood MT, Evans DA, , 1997. Cutaneous leishmaniasis: iso-enzyme characterisation of Leishmania tropica . J Pak Med Assoc 47: 270273. [Google Scholar]
  8. Rowland M, Munir A, Durrani N, Noyes H, Reyburn H, , 1999. An outbreak of cutaneous leishmaniasis in an Afghan refugee settlement in northwest Pakistan. Trans R Soc Trop Med Hyg 93: 133136.[Crossref] [Google Scholar]
  9. Noor SM, Hussain D, , 2004. Cutaneous leishmaniasis in Sadda, Kurram agency, Pakistan. J Pak Assoc Dermatol 14: 114117. [Google Scholar]
  10. WHO, 2001. Afghanistan Crisis-Special Report: Leishmaniasis in Pakistan. Geneva, Switzerland: World Health Organization. [Google Scholar]
  11. Afghan AK, Kassi M, Kasi PM, Ayub A, Kakar N, Marri SM, , 2011. Clinical manifestations and distribution of cutaneous leishmaniasis in Pakistan. J Trop Med 2011: 359145.[Crossref] [Google Scholar]
  12. Bhutto AM, Soomro RA, Nonaka S, Hashiguchi Y, , 2003. Detection of new endemic areas of cutaneous leishmaniasis in Pakistan: a 6-year study. Int J Dermatol 42: 543548.[Crossref] [Google Scholar]
  13. Bari AU, , 2006. Epidemiology of cutaneous leishmaniasis. J Pak Assoc Dermatol 16: 156162. [Google Scholar]
  14. Bari AU, , 2012. Clinical spectrum of cutaneous leishmaniasis: an overview from Pakistan. Dermatol Online J 18: 4. [Google Scholar]
  15. Rahman S, Abdullah FH, Khan JA, , 2009. The frequency of old world cutaneous leishmaniasis in skin ulcers in Peshawar. J Ayub Med Coll Abbottabad 21: 7275. [Google Scholar]
  16. Raja KM, Khan AA, Hameed A, Rahman SB, , 1998. Unusual clinical variants of cutaneous leishmaniasis in Pakistan. Br J Dermatol 139: 111113.[Crossref] [Google Scholar]
  17. Bari AU, Rahman SB, , 2008. Many faces of cutaneous leishmaniasis. Indian J Dermatol Venereol Leprol 74: 2327.[Crossref] [Google Scholar]
  18. Bari AU, Raza N, , 2010. Lupoid cutaneous leishmaniasis: a report of of 16 cases. Indian J Dermatol Venereol 76: 85.[Crossref] [Google Scholar]
  19. Shamsuddin S, Mengal JA, Gazozai S, Mandokhail ZK, Kasi M, Muhammad N, , 2006. Atypical presentation of cutaneous leishmaniasis in native population of Baluchistan. J Pak Assoc Dermatol 16: 196200. [Google Scholar]
  20. Plowe CV, Djimde A, Bouare M, Doumbo O, Wellems TE, , 1995. Pyrimethamine and proguanil resistance-conferring mutations in Plasmodium falciparum dihydrofolate reductase: polymerase chain reaction methods for surveillance in Africa. Am J Trop Med Hyg 52: 565568. [Google Scholar]
  21. Parvizi P, Mauricio I, Aransay AM, Miles MA, Ready PD, , 2005. First detection of Leishmania major in peridomestic Phlebotomus papatasi from Isfahan province, Iran: comparison of nested PCR of nuclear ITS ribosomal DNA and semi-nested PCR of minicircle kinetoplast DNA. Acta Trop 93: 7583.[Crossref] [Google Scholar]
  22. Bulle B, Millon L, Bart J-M, Gallego M, Gambarelli F, Portus M, Schnur L, Jaffe CL, Fernandez-Barredo S, Alunda JM, Piarroux R, , 2002. Practical approach for typing strains of Leishmania infantum by microsatellite analysis. J Clin Microbiol 40: 33913397.[Crossref] [Google Scholar]
  23. Parikh R, Mathai A, Parikh S, Chandra Sekhar G, Thomas R, , 2008. Understanding and using sensitivity, specificity and predictive values. Indian J Ophthalmol 56: 4550.[Crossref] [Google Scholar]
  24. Bensoussan E, Nasereddin A, Jonas F, Schnur LF, Jaffe CL, , 2006. Comparison of PCR assays for diagnosis of cutaneous leishmaniasis. J Clin Microbiol 44: 14351439.[Crossref] [Google Scholar]
  25. Chargui N, Bastien P, Kallel K, Haouas N, Akrout FM, Masmoudi A, Zili J, Chaker E, Othman AD, Azaiez R, Crobu L, Mezhoud H, Babba H, , 2005. Usefulness of PCR in the diagnosis of cutaneous leishmaniasis in Tunisia. Trans R Soc Trop Med Hyg 99: 762768.[Crossref] [Google Scholar]
  26. Weigle KA, Labrada LA, Lozano C, Santrich C, Barker DC, , 2002. PCR-based diagnosis of acute and chronic cutaneous leishmaniasis caused by Leishmania (Viannia). J Clin Microbiol 40: 601606.[Crossref] [Google Scholar]
  27. Khan Z, , 2005. Cutaneous leishmaniasis in N.W.F.P. J Postgrad Med Inst 19: 226228. [Google Scholar]
  28. Reithinger R, Mohsen M, Aadil K, Sidiqi M, Erasmus P, Coleman PG, , 2003. Anthroponotic cutaneous leishmaniasis, Kabul, Afghanistan. Emerg Infect Dis 9: 727729.[Crossref] [Google Scholar]
  29. Bashaye S, Nombela N, Argaw D, Mulugeta A, Herrero M, Nieto J, Chicharro C, Canavate C, Aparicio P, Velez ID, Alvar J, Bern C, , 2009. Risk factors for visceral leishmaniasis in a new epidemic site in Amhara Region, Ethiopia. Am J Trop Med Hyg 81: 3439. [Google Scholar]
  30. Mengesha B, Endris M, Takele Y, Mekonnen K, Tadesse T, Feleke A, Diro E, , 2014. Prevalence of malnutrition and associated risk factors among adult visceral leishmaniasis patients in northwest Ethiopia: a cross sectional study. BMC Res Notes 7: 75.[Crossref] [Google Scholar]
  31. Kassi M, Afghan AK, Rehman R, Kasi PM, , 2008. Marring leishmaniasis: the stigmatization and the impact of cutaneous leishmaniasis in Pakistan and Afghanistan. PLoS Negl Trop Dis 2: e259.[Crossref] [Google Scholar]
  32. Brooker S, Mohammed N, Adil K, Agha S, Reithinger R, Rowland M, Ali I, Kolaczinski J, , 2004. Leishmaniasis in refugee and local Pakistani populations. Emerg Infect Dis 10: 16811684.[Crossref] [Google Scholar]
  33. Kolaczinski J, Brooker S, Reyburn H, Rowland M, , 2004. Epidemiology of anthroponotic cutaneous leishmaniasis in Afghan refugee camps in northwest Pakistan. Trans R Soc Trop Med Hyg 98: 373378.[Crossref] [Google Scholar]
  34. Ramirez JR, Agudelo S, Muskus C, Alzate JF, Berberich C, Barker D, Velez ID, , 2000. Diagnosis of cutaneous leishmaniasis in Colombia: the sampling site within lesions influences the sensitivity of parasitologic diagnosis. J Clin Microbiol 38: 37683773. [Google Scholar]
  35. Uzun S, Durdu M, Culha G, Allahverdiyev AM, Memisoglu HR, , 2004. Clinical features, epidemiology, and efficacy and safety of intralesional antimony treatment of cutaneous leishmaniasis: recent experience in Turkey. J Parasitol 90: 853859.[Crossref] [Google Scholar]
  36. Singh S, Dey A, Sivakumar R, , 2005. Applications of molecular methods for Leishmania control. Expert Rev Mol Diagn 5: 251265.[Crossref] [Google Scholar]
  37. Herwaldt BL, , 1999. Leishmaniasis. Lancet 354: 11911199.[Crossref] [Google Scholar]
  38. Reithinger R, Dujardin JC, , 2007. Molecular diagnosis of leishmaniasis: current status and future applications. J Clin Microbiol 45: 2125.[Crossref] [Google Scholar]
  39. Boggild AK, Valencia BM, Espinosa D, Veland N, Ramos AP, Arevalo J, Llanos-Cuentas A, Low DE, , 2010. Detection and species identification of Leishmania DNA from filter paper lesion impressions for patients with American cutaneous leishmaniasis. Clin Infect Dis 50: e1e6.[Crossref] [Google Scholar]
  40. da Silva ES, Gontijo CM, Pacheco R da S, Brazil RP, , 2004. Diagnosis of human visceral leishmaniasis by PCR using blood samples spotted on filter paper. Genet Mol Res 3: 251257. [Google Scholar]
  41. Miranda A, Saldana A, Gonzalez K, Paz H, Santamaria G, Samudio F, Calzada JE, , 2012. Evaluation of PCR for cutaneous leishmaniasis diagnosis and species identification using filter paper samples in Panama, Central America. Trans R Soc Trop Med Hyg 106: 544548.[Crossref] [Google Scholar]
  42. Ovalle Bracho C, Porras de Quintana L, Muvdi Arenas S, Rios Parra M, , 2007. Polymerase chain reaction with two molecular targets in mucosal leishmaniasis' diagnosis: a validation study. Mem Inst Oswaldo Cruz 102: 549554.[Crossref] [Google Scholar]
  43. de Almeida ME, Steurer FJ, Koru O, Herwaldt BL, Pieniazek NJ, da Silva AJ, , 2011. Identification of Leishmania spp. by molecular amplification and DNA sequencing analysis of a fragment of rRNA internal transcribed spacer 2. J Clin Microbiol 49: 31433149.[Crossref] [Google Scholar]
  44. Schonian G, Nasereddin A, Dinse N, Schweynoch C, Schallig HDFH, Presber W, Jaffe CL, , 2003. PCR diagnosis and characterization of Leishmania in local and imported clinical samples. Diagn Microbiol Infect Dis 47: 349358.[Crossref] [Google Scholar]
  45. Ayub S, Gramiccia M, Khalid M, Mujtaba G, Bhutta RA, , 2003. Cutaneous leishmaniasis in Multan: species identification. J Pak Med Assoc 53: 445447. [Google Scholar]
  46. Myint CK, Asato Y, Yamamoto Y-i, Kato H, Bhutto AM, Soomro FR, Memon MZ, Matsumoto J, Marco JD, Oshiro M, Katakura K, Hashiguchi Y, Uezato H, , 2008. Polymorphisms of cytochrome b gene in Leishmania parasites and their relation to types of cutaneous leishmaniasis lesions in Pakistan. J Dermatol 35: 7685.[Crossref] [Google Scholar]
  47. Bhutto AM, Soomro FR, Baloch JH, Matsumoto J, Uezato H, Hashiguchi Y, Katakura K, , 2009. Cutaneous leishmaniasis caused by Leishmania (L.) major infection in Sindh province, Pakistan. Acta Trop 111: 295298.[Crossref] [Google Scholar]
  48. Marco JD, Bhutto AM, Soomro FR, Baloch JH, Barroso PA, Kato H, Uezato H, Katakura K, Korenaga M, Nonaka S, Hashiguchi Y, , 2006. Multilocus enzyme electrophoresis and cytochrome B gene sequencing-based identification of Leishmania isolates from different foci of cutaneous leishmaniasis in Pakistan. Am J Trop Med Hyg 75: 261266. [Google Scholar]
  49. Shakila A, Bilqees FM, Salim A, Moinuddin M, , 2006. Geographical distribution of cutaneous leishmaniasis and sand flies in Pakistan. Turkiye Parazitol Derg 30: 16. [Google Scholar]
  50. Rab MA, Iqbal J, Azmi FH, Munir MA, Saleem M, , 1989. Visceral leishmaniasis: a seroepidemiological study of 289 children from endemic foci in Azad Jammu and Kashmir by indirect fluorescent antibody technique. J Pak Med Assoc 39: 225228. [Google Scholar]
  51. Rahim F, Rehman F, Ahmad S, Zada B, , 1998. Visceral leishmaniasis in District Dir, NWFP. J Pak Med Assoc 48: 161162. [Google Scholar]
  52. Rab MA, Evans DA, , 1995. Leishmania infantum in the Himalayas. Trans R Soc Trop Med Hyg 89: 2732.[Crossref] [Google Scholar]
  53. Momeni AZ, Aminjavaheri M, , 1994. Clinical picture of cutaneous leishmaniasis in Isfahan, Iran. Int J Dermatol 33: 260265.[Crossref] [Google Scholar]
  54. Bari AU, Hasshim R, Mahmood K, Muhammad I, Shahbaz N, Tariq KM, , 2011. Clinico-epidemiological pattern of cutaneous leishmaniasis in armed forces personnel fighting war against terrorism in Khyber Pakhtunkhwa Province and Fata regions. J Pak Assoc Dermatol 21: 1015. [Google Scholar]
  55. Reithinger R, Mohsen M, Wahid M, Bismullah M, Quinnell RJ, Davies CR, Kolaczinski J, David JR, , 2005. Efficacy of thermotherapy to treat cutaneous leishmaniasis caused by Leishmania tropica in Kabul, Afghanistan: a randomized, controlled trial. Clin Infect Dis 40: 11481155.[Crossref] [Google Scholar]
  56. Klaus SN, Frankenburg S, Dhar AD, Freedberg IM, Eisen AZ, Wolff K, Austen KF, Goldsmith LA, Si K, , 2003. Leishmaniasis and other protozoan infections. , eds. Fitzpatrick's Dermatology in General Medicine, 6th edition. New York, NY: McGraw-Hill, 22152221. [Google Scholar]
  57. Pearson RD, Sousa ADQ, Jeronimo SMB, Mandell GL, Bennett JE, Dolin R, , 2000. Leishmania species: visceral (Kala-Azar), cutaneous, and mucosal leishmaniasis. , eds. Mandell, Douglas and Bennett's Principles and Practice of Infectious Disease, 5th edition, Vol. 2. Philadelphia, PA: Churchill Livingstone, 28312846. [Google Scholar]
  58. Mujtaba G, Khalid M, , 1998. Cutaneous leishmaniasis in Multan, Pakistan. Int J Dermatol 37: 843845.[Crossref] [Google Scholar]
  59. Bari AU, Ejaz A, , 2009. Fissure leishmaniasis: a new variant of cutaneous leishmaniasis. Dermatol Online J 15: 13. [Google Scholar]

Data & Media loading...

Supplementary PDF

  • Received : 30 Apr 2016
  • Accepted : 22 Jul 2016
  • Published online : 02 Nov 2016

Most Cited This Month

This is a required field
Please enter a valid email address
Approval was a Success
Invalid data
An Error Occurred
Approval was partially successful, following selected items could not be processed due to error