Türkiyenin Çukurova Bölgesinde Kliniksel Materyalde Bulunan Leishmania Parazitlerinin Polimeraz Zincir Reaksiyonu Ile Araştırılması

Abstract

Giriş: Leishmania cinsi parazitlerinin sebep olduğu Leishmaniazis, oldukça önemli bir sağlık problemi olduğu düşünülür. Klinik materyalde Leishmania parazitlerinin belirlenmesi ve tür ayırımında polimeraz zincir reaksiyonurestriksiyon fragman uzunluğu polimorfizm metodunu PCR-RFLP kullanmayı ve mikroskobik inceleme sonuçlarıyla karşılaştırmayı amaçladık. Metotlar: Kırk beş deri lezyonu, 9 kemik iliği ve 4 referans türünden oluşan toplam 58 örnek, bu çalışmaya alındı. Örnekler hem DNA elde edilmesi ve hem de preparat hazırlanmasında kullanıldı. DNA’lar, Leishmania alt-cinsinin belirlenmesi için PCR yöntemi ile çoğaltıldı ve PCR ürünü, tür ayırımı için HaeIII ile kesildi. Sonuçlar: Visseral Leishmania parazitleri Leishmania infantum olarak belirlendi. Deri leişmanyazın 45 hastanın, çoğunda Leishmania tropica belirlenirken, diğerleri Leishmania donovani’ye benzer bulundu. Aynı zamanda PCR’ın pozitiflik oranı %100 , mikroskobik inceleme %67 sonuçlarından daha fazla olduğu saptandı. Çıkarım: PCR-RFLP tekniği, Leishmania türlerinin tespit ve ayırımında çok hassas olduğu gözlendi

Keywords

• Tipleme,Leishmaniazis,PCR,RFLP

Investigation Of Leishmania Parasites From Clinical Samples Using Polymerase Chain Reaction Technique In Cukurova Region, Turkey

Abstract

Background: Leishmaniasis, caused by parasites of the genus Leishmania is still considered an important health problem. We aimed to appraise a Polymerase Chain Reaction-Restriction Fragment Length Polymorphism PCR-RFLP method for the detection and species differentiation of Leishmania parasites in clinical samples; and to compare with the results of microscopic examination. Methods: Totally 58 samples were taken from 45 skin lesion, nine bone marrow aspirates, and four reference strains. The samples were used for both DNA and smear-slide preparations. The DNAs were amplified by PCR for the detection of Leishmania subgenus and PCR products were restricted with HaeIII for the species differentiation. Results: The visceral Leishmania parasites VL were genotyped as Leishmania infantum. Of 45 patients with CL, most of them were analyzed as Leishmania tropica, but the others were characterized similar to Leishmania donovani reference. It was also determined positivity rate in PCR 100% was higher than microscopic examination about 67% . Conclusions: PCR-RFLP technique appears to be most sensitive for the detection and the differentiation of Leishmania species

Keywords

• Identification, Leishmaniasis, PCR, RFLP.

References

1. Uzun S, Uslular C, Yucel A, Acar MA, Ozpoyraz M, Memisoglu HR. Cutaneous leishmaniasis: evaluation of 3,074 cases in the Cukurova region of Turkey. Br J Dermatol 1999; 140:347-350.
2. Dilmec F, Matur A, Uzun S, Karakas M, Memisoglu HR. Comparision of leishmania sp. DNA’s isolated in the regions of Çukurova and Şanlıurfa with the restriction endonucleases. Journal of Harran University Medical Faculty 2004; 1:21-27.
3. Dillon DC, Day CH, Whittle JA, Magill AJ, Reed SG. Characterization of a Leishmania tropica antigen that detects immune responses in Desert Storm viscerotropic leishmaniasis patients. Proc Natl Acad Sci USA 1995; 92:7981-7985.
4. Ben-Ami R, Schnur LF, Golan Y, Jaffe CL, Mardi T, Zeltser D. Cutaneous involvement in a rare case of adult visceral leishmaniasis acquired in Israel. J Infect 2002; 44:181-184.
5. del Giudice P, Marty P, Lacour JP, Perrin C, Pratlong F, Haas H, et al. Cutaneous leishmaniasis due to Leishmania infantum. Case reports and literature review. Arch Dermatol 1998; 134:193-198.
6. Elamin EM, Guizani I, Guerbouj S, Gramiccia M, El Hassan AM, Di Muccio T, et al. Identification of Leishmania donovani as a cause of cutaneous leishmaniasis in Sudan. Trans R Soc Trop Med Hyg 2008; 102:54-57 [Epub ahead of print].
7. Serin MS, Daglioglu K, Bagirova M, Allahverdiyev A, Uzun S, Vural Z, et al. Rapid Leishmania isolates from cutaneous and visceral leishmaniasis by microcapillary cultivation and polymerase chain reaction- restriction fragment length polymorphism of miniexon region. Diagn Microbiol Infect Dis 2005; 53: 209-214. genotyping of
8. Singh B. Molecular methods for diagnosis and epidemiological studies of parasitic infections. Int J Parasitol 1997; 27:1135- 1145.

9. Andresen K, Gaafar A, El-Hassan AM, Ismail A, Dafalla M, Theander TG, et al. Evaluation of the polymerase chain reaction in the diagnosis of cutaneous leishmaniasis due to Leishmania major a comparison with direct microscopy of smears and sections from lesions. Trans R Soc Trop Med Hyg 1996; 90:133-135.
10. Culha G, Uzun S, Ozcan K, Memisoglu HR, Chang KP. Comparison of conventional and polymerase techniques for leishmaniasis in the endemic region of Adana, Turkey. Int J Dermatol 2006; 45:569-572. diagnostic
11. Chargui N, Bastien P, Kallel K, Haouas N, Akrout FM, Masmoudi A, et al. Usefulness of PCR in the diagnosis of cutaneous leishmaniasis in Tunisia. Trans R Soc Trop Med Hyg 2005; 99:762-768.
12. Mathis A, Deplazes P. PCR and In Vitro Cultivation for Detection of Leishmania spp.in Diagnostic Samples from Humans and Dogs. J Clin Microbiol 1995; 33:1145-1149.
13. Minodier P, Piarroux R, Gambarelli F, Joblet C, Dumon H. Rapid identification of causative species in patients with Old World leishmaniasis. J Clin Microbiol 1997; 35:2551-2555.
14. Schönian G, Nasereddin A, Dinse N, Schweynoch C, Schallig HD, Presber W, et al. PCR diagnosis and characterization of Leishmania in local and imported clinical samples. Diagn Microbiol Infect Dis 2003; 47:349-358.
15. Mahboudi F, Abolhassan M, Yaran M, Mobtaker H, Azizi M. Identification and Differentiation of Iranian Leishmania Species by PCR Amplificiation of kDNA. Scand J Infect Dis 2001; 33:596-598.
16. Marfurt J, Nasereddin A, Niederwieser I, Jaffe CL, Beck HP, Felger I. Identification and differentiation of Leishmania species in clinical samples by PCR amplification of the miniexon restriction fragment length polymorphism analysis. J Clin Microbiol 2003; 41:3147- 3153. and subsequent
17. Knio KN, Baydoun E, Tawk R, Nuwayri- Salti N. Isoenzyme characterization of Leishmania isolates from Lebanon and Syria. Am J Trop Med Hyg 2000; 63:43-77.
18. Ozbel Y, Oskam L, Ozensoy S, Turgay N, Alkan MZ, Jaffe CL, et al. A survey on canine leishmaniasis in western Turkey by parasite, DNA and antibody detection assays. Acta Trop 2000; 74:1-6.