Mikroskop incelemesi negatif olan şüpheli kronik kutanöz leishmania olgularının polimeraz zincir reaksiyonu yöntemi ile araştırılması
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Tarih
2019
Dergi Başlığı
Dergi ISSN
Cilt Başlığı
Yayıncı
Ankara Mikrobiyoloji Derneği
Erişim Hakkı
info:eu-repo/semantics/openAccess
Özet
Leyşmanyazis, enfekte dişi tatarcıkların ısırmasıyla insanlara bulaşan paraziter bir hastalıktır. Kutanöz
leyşmanyazisin (KL) tanısında dermal kazıntıdan alınan örneklerde, mikroskopta parazitin gösterilmesi
altın standart bir yöntem olarak yerini korumaktadır. Ancak bir yıl ve daha uzun süreli lezyonu bulunan
kronik olgularda amastigot sayısı çok az sayıda olduğundan saptamak zordur. Hastaların öncelikle vücutlarında çıkan bu lezyonları dikkate almamaları, sağlık kurumlarına başvurmamaları veya geç başvurmaları,
yanlış tedavi almaları gibi birçok nedenden ötürü tanı ve tedavi gecikmektedir. Ayrıca, lezyonlar üzerine
sekonder enfeksiyon eklenerek prognoz kötüleşmekte ve yaralar kronikleşmektedir. Bu nedenle, kronik
KL şüpheli olgularda mikroskop ile inceleme yöntemine ek olarak moleküler yöntemlerden faydalanılmaktadır. Bu çalışmada, mikroskop incelemesi sonucunda Leishmania amastigotları görülmeyen ancak
klinik olarak KL açısından değerlendirilmesi rapor edilmiş şüpheli kronik KL Türkiye başlangıçlı olgulara ait
dermal kazıntı preparatları seçilerek polimeraz zincir reaksiyonu (PCR) yöntemi kullanılarak leyşmanyazis
tanısında moleküler yöntemin tanı değerinin ortaya konması amaçlanmıştır. Hatay Mustafa Kemal Üniversitesi Tıp Fakültesi Parazitoloji Laboratuvarına farklı polikliniklerden gelen KL şüphesi ile başvurmuş ve mikroskop incelemesi sonucunda endemik bölgelerden geldikleri kaydedilmiş (Hassa, Altınözü, Yayladağı gibi), klinik olarak kronik KL açısından değerlendirilmesi rapor edilmiş Türk hastaya ait dermal kazıntı preparatları (n= 50) çalışmaya dahil edilmiştir. Örneklerden DNA izolasyonu yapılarak tüm Leishmania türlerine özgü kinetoplastit DNA (kDNA) bölgesini hedefleyen 13A, 13B primerleri kullanılmıştır. PCR ile pozitif bulunan örnekler internal transcribed spacer (ITS-1) bölgesini hedefleyen LITSR ve L5.8S primerleri kullanılarak “polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP)” yöntemi ile tür tayini yapılmıştır. Toplam 50 adet dermal kazıntı örneğinden 17 (%34)’si kDNA bölgesini hedefleyen 13A, 13B primerleri ile pozitif olarak bulunmuştur. Pozitif bulunan örnekler ITS-1 bölgesini hedefleyen LITSR ve L5.8S primerleri ile de pozitif olarak saptanmıştır. ITS-1 gen bölgesiyle yapılan PCR sonucunda elde edilen ürünler BsuRI (HaeIII) enzimi kullanılarak kesilmiştir. PCR-RFLP analizi sonucunda 17 örneğin 11’i Leishmania tropica, biri Leishmania major ve beşi Leishmania infantum/donovani olarak tanımlanmıştır.
Kronik KL; sarkoidoz, tüberküloz, malign tümörler gibi deri hastalıklarıyla karışabilmektedir. Özellikle,
kronik KL olguları doğru tanı konulamaması, mikroskop deneyiminin yeterli olmaması, parazit sayısının
az olması nedeniyle görülememesi gibi pek çok sebepten gözden kaçabilmektedir. Bu nedenle kronik KL
şüphesi olan ancak mikroskop inceleme yöntemi ile parazit saptanamayan örneklerde moleküler yöntem
olan PCR’nin kullanılmasının gerektiği sonucuna varılmıştır
Leishmaniasis is a parasitic disease that is transmitted to humans by the bites of infected female phlebotomine sandflies. In the diagnosis of cutaneous leishmaniasis (CL), in the smear samples, the demonstration of the parasite by microscope remains a gold standard method. However, it becomes difficult to diagnose the parasite since the number of amastigotes in chronic cases with a lesion of one year or longer is very low. Due to many factor such as patients primarily do not to take any notice these lesions in their bodies, do not apply to health institutions or late applied, receive wrong treatment; the diagnosis and treatment are delayed. In addition, it is been worse prognosis by add secondary infection to lesions and wounds become chronic. For this reason, molecular methods are used in addition to microscopic examination in chronic suspected CL cases. It was aimed to reveal of the molecular diagnostic value in chronic suspected CL cases by polymerase chain reaction (PCR) in the smear belonging to Turkish patients that reported to be evaluated clinically because it can not be seen Leishmania amastigotes in microscopic examination. Smear of 50 Turkish patients who were clinically reported of the evaluation of chronic CL were selected. These samples were smears belonging to suspected CL patients that applied Hatay Mustafa Kemal University, Faculty of Medicine, Parasitology laboratory from different polyclinics and were decided to be evaluated clinically as a result of microscopic examination because they came from endemic regions (such as Hassa, Altınözü, Yayladağı). DNA was isolated from selected samples and PCR was performed using 13A, 13B primers targeting the kinetoplastid DNA (kDNA) region. The samples found positive by PCR were typed by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis using LITSR and L5.8S primers targeting internal transcribed spacer (ITS-1) region. Of the 50 smear samples, 17 (34%) were determined positive with 13A, 13B primers targeting the kinetoplastid DNA (kDNA) region. Positive samples were also found to be positive with LITSR and L5.8S primers targeting ITS-1 region. The PCR products obtained from PCR with ITS-1 gene region were digested with the restriction endonucleases BsuRI (HaeIII). As a result of PCR-RFLP analysis, it was determined that 11 of Leishmania tropica, one of Leishmania major and five of Leishmania infantum/donovani out of 17 samples. Chronic CL can be confused with skin diseases such as sarcoidosis, tuberculosis, malignant tumors. In particular, chronic CL cases can be escaped the attention for many reasons such as failure to diagnose correctly, insufficient microscope experience, fail to see due to low number of parasites. For this reason, it was concluded that PCR, which is a molecular method, should be used in chronic suspected CL samples which are negative for the parasite by microscopic examination.
Leishmaniasis is a parasitic disease that is transmitted to humans by the bites of infected female phlebotomine sandflies. In the diagnosis of cutaneous leishmaniasis (CL), in the smear samples, the demonstration of the parasite by microscope remains a gold standard method. However, it becomes difficult to diagnose the parasite since the number of amastigotes in chronic cases with a lesion of one year or longer is very low. Due to many factor such as patients primarily do not to take any notice these lesions in their bodies, do not apply to health institutions or late applied, receive wrong treatment; the diagnosis and treatment are delayed. In addition, it is been worse prognosis by add secondary infection to lesions and wounds become chronic. For this reason, molecular methods are used in addition to microscopic examination in chronic suspected CL cases. It was aimed to reveal of the molecular diagnostic value in chronic suspected CL cases by polymerase chain reaction (PCR) in the smear belonging to Turkish patients that reported to be evaluated clinically because it can not be seen Leishmania amastigotes in microscopic examination. Smear of 50 Turkish patients who were clinically reported of the evaluation of chronic CL were selected. These samples were smears belonging to suspected CL patients that applied Hatay Mustafa Kemal University, Faculty of Medicine, Parasitology laboratory from different polyclinics and were decided to be evaluated clinically as a result of microscopic examination because they came from endemic regions (such as Hassa, Altınözü, Yayladağı). DNA was isolated from selected samples and PCR was performed using 13A, 13B primers targeting the kinetoplastid DNA (kDNA) region. The samples found positive by PCR were typed by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis using LITSR and L5.8S primers targeting internal transcribed spacer (ITS-1) region. Of the 50 smear samples, 17 (34%) were determined positive with 13A, 13B primers targeting the kinetoplastid DNA (kDNA) region. Positive samples were also found to be positive with LITSR and L5.8S primers targeting ITS-1 region. The PCR products obtained from PCR with ITS-1 gene region were digested with the restriction endonucleases BsuRI (HaeIII). As a result of PCR-RFLP analysis, it was determined that 11 of Leishmania tropica, one of Leishmania major and five of Leishmania infantum/donovani out of 17 samples. Chronic CL can be confused with skin diseases such as sarcoidosis, tuberculosis, malignant tumors. In particular, chronic CL cases can be escaped the attention for many reasons such as failure to diagnose correctly, insufficient microscope experience, fail to see due to low number of parasites. For this reason, it was concluded that PCR, which is a molecular method, should be used in chronic suspected CL samples which are negative for the parasite by microscopic examination.
Açıklama
Anahtar Kelimeler
Kronik şüpheli leyşmanyazis, Tanı, Polimeraz zincir reaksiyonu, Restriction fragment length polymorphism, Chronic suspected leishmaniasis, Diagnosis, Polymerase chain reaction, Restriction fragment length polymorphism
Kaynak
Mikrobiyoloji Bülteni
WoS Q Değeri
Q4
Scopus Q Değeri
Q3
Cilt
53
Sayı
4
Künye
ÇULHA G,KAYA T,DURAN G. G,KÜÇÜK M. U,DOĞRAMACI A. Ç,ÇELİK D. T (2019). Mikroskop İncelemesi Negatif Olan
Şüpheli Kronik Kutanöz Leishmania Olgularının Polimeraz Zincir Reaksiyonu Yöntemi ile Araştırılması. Mikrobiyoloji Bülteni, 53(4), 408 - 418. Doi: 10.5578/mb.68692
