Kala-azarlı hastalarda etken türlerin PCR-RFLP yöntemi ile tanımlanması
Identification of causative species in patients with visceral leishmaniosis by PCR-RFLP assay
Yazar: DİLEK NAS ÖZERDEM
Danışman: PROF. DR. İSMAİL SONER KOLTAŞ
Çukurova Üniversitesi / Sağlık Bilimleri Enstitüsü / Parazitoloji Ana Bilim Dalı
Derece: Doktora, 2009
Konu: Parazitoloji = Parasitology
Dizin: Leishmania = Leishmania ; Leishmaniasis = Leishmaniasis ; Leishmaniasis-visceral = Leishmaniasis-visceral ; Polimeraz zincirleme reaksiyonu = Polymerase chain reaction ; Teşhis = Diagnosis ; Teşhis-ayırıcı = Diagnosis-differential

Özet:
Kala azar( VL) özellikle Çukurova bölgesinde ve ülkemizde halen önemli bir halk sağlığı sorunudur. Akdeniz'e kıyısı bulunan ülkelerde endemiktir. Hastalığın tanısında kemik iliğinden ve kandan yapılan yayma preparat, kültür ve serolojik yöntemler kullanılmaktadır. Visseral leyişmanyoz (VL) endemik bölgelerde yaşayan immun sistemi baskılanmış HIV enfeksiyonlu hastalarda olduğu gibi bağışıklığı yetersiz hastalarda da fırsatçı bir infeksiyon olarak bildirilmektedir. Bu yüzden visseral leyişmanyozun tanısı böyle hastalarda zordur, serolojik tanı güvenilir sonuçlar vermemekte, ayrıca kültür yöntemi ve doğrudan mikroskop bakısı da hassas olmamakta ve kültür zaman alıcı olmaktadır. Bu nedenle, son yıllarda hastalığın tanısı ve daha da önemlisi hangi türün hastalığa neden olduğunu saptamak için özellikle DNA'ya dayalı moleküler yöntemler geliştirilmiştir.Bu çalışmada visseral leyişmanyoz ön tanısı ile izlenen 50 hastaya ait kan örnekleri doğrudan mikroskop bakısı, rK39 hızlı tanı testi ve PCR-RFLP yöntemi ile çalışıldı. On hastanın doğrudan mikroskop bakısında amastigotlar görülürken, 40 hastanın mikroskop bakısında amastigotlar görülmedi. On üç hasta rK39 (+) pozitif, otuz yedi hasta rK39(-) negatif bulundu. Yirmi dokuz hastanın PCR (+) pozitif, yirmi bir hastanın PCR (-) negatif olduğu görüldü. PCR' ın doğrudan mikroskop bakısı ve rK39 hızlı tanı testine oranla sırasıyla %90 -% 100 duyarlı ve %50-% 56.8 özgül olduğu görüldü. Sonuç olarak bu çalışmada etken türlerin L.infantum ve L.donovani olduğu saptandı.Anahtar Sözcükler: Visseral leyişmanyoz, ayırıcı tanı, PCR-RFLP.ABSTRACTIDENTIFICATION OF CAUSATIVE SPECIES IN PATIENTS WITH VISCERAL LEISHMANIOSIS BY PCR-RFLP ASSAYVisceral leishmaniosis is a health care problem especially in Çukurova region and in Turkey. Visceral leishmaniosis is endemic in the countries beside of the Meditterranean Sea. Direct microscopic examination from blood and bone marrow, culture and serological tests are the methods that used for diagnosis of visceral leishmaniosis. Visceral leishmaniosis has been reported to be an oppurtunistic infection in immunosuppressed patients and immunocompromised subjects such as patients with human immunodeficiency virus infections living in areas in which visceral leishmaniosis is endemic. Because of this the diagnosis of visceral leishmaniosis is difficult in such patients, serological diagnosis is not reliable for immunosuppressed patients, direct diagnosis and culture is not sensitive and culture is time consuming. Therefore recently several methods especially based on DNA have been developed for the diagnosis and more important from that for the identification of the species which caused infections.In this study, blood samples belonging to 50 patients whose were following by preliminary diagnosis of visceral leishmaniosis were studied by direct microscopic examination, rK39 dipstick test and PCR-RFLP. Amastigote forms were detected from 10 samples, and not detected other 40 samples by direct microscopic examination. Thirteen samples were found to be rK39 (+) and 37 samples were found to be rK39 (-). Twenty nine samples were (+) and 21 samples were (-) by PCR. Sensitivity of PCR was found to be %90-%100 compare to direct microscopic examination and rK39 dipstick test. On the other side specifity of PCR was found to be %50-%56.8 compare to direct microscopic examination and rK39 dipstick test. As a result, in this study we found the causative species as L.infantum and L.donovani.Key Words: Visceral leishmaniosis, identification, PCR-RFLP.

Abstract:
Visceral leishmaniosis is a health care problem especially in Çukurova region and in Turkey. Visceral leishmaniosis is endemic in the countries beside of the Meditterranean Sea. Direct microscopic examination from blood and bone marrow, culture and serological tests are the methods that used for diagnosis of visceral leishmaniosis. Visceral leishmaniosis has been reported to be an oppurtunistic infection in immunosuppressed patients and immunocompromised subjects such as patients with human immunodeficiency virus infections living in areas in which visceral leishmaniosis is endemic. Because of this the diagnosis of visceral leishmaniosis is difficult in such patients, serological diagnosis is not reliable for immunosuppressed patients, direct diagnosis and culture is not sensitive and culture is time consuming. Therefore recently several methods especially based on DNA have been developed for the diagnosis and more important from that for the identification of the species which caused infections.In this study, blood samples belonging to 50 patients whose were following by preliminary diagnosis of visceral leishmaniosis were studied by direct microscopic examination, rK39 dipstick test and PCR-RFLP. Amastigote forms were detected from 10 samples, and not detected other 40 samples by direct microscopic examination. Thirteen samples were found to be rK39 (+) and 37 samples were found to be rK39 (-). Twenty nine samples were (+) and 21 samples were (-) by PCR. Sensitivity of PCR was found to be %90-%100 compare to direct microscopic examination and rK39 dipstick test. On the other side specifity of PCR was found to be %50-%56.8 compare to direct microscopic examination and rK39 dipstick test. As a result, in this study we found the causative species as L.infantum and L.donovani.Key Words: Visceral leishmaniosis, identification, PCR-RFLP.