Türkiye'de kronik kutanöz leishmaniasis'de in vitro ve in vivo modelde miltefosin etkinliğinin değerlendirilmesi
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Dosyalar
Tarih
2018
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Yayıncı
Ege Üniversitesi, Sağlık Bilimleri Enstitüsü
Erişim Hakkı
info:eu-repo/semantics/openAccess
Özet
Leishmaniasis, Dünya Sağlık Örgütü'nün (DSÖ) ihmal edilen hastalıklar listesinde bulunan ve Dünya'nın tropikal ve subtropikal bölgelerinde farklı kliniklerde enfeksiyonlara sebep olan vektör kaynaklı bir protozoon hastalığıdır. Dünyada milyonlarca kişide enfeksiyona neden olan leishmaniasisin üç temel klinik formu bulunmaktadır; iç organları tutarak karakterize olan formu visseral leishmaniasis (VL), deri tutulumu ile lezyonlara sebep olan kutanöz leishmaniasis (KL) ve mukoza ve deriyi beraber etkileyen muko-kutanöz leishmaniasis (MKL). Ateş veya genel semptomlara neden olmadan, genellikle bir veya birden fazla uzun süreli deri lezyonu ile seyreden KL, temelde iki büyük gruba ayrılabilir; akut KL (AKL) ve kronik KL (KKL). KL lezyonları 2 yıl içinde tedavi ile veya kendiliğinden iyileşmez ise KKL olarak adlandırılmaktadır. KL, Türkiye'de 1980'li yıllardan beri ihbarı zorunlu hastalıklar arasında yer almaktadır. Sağlık Bakanlığı verilerine göre 2005-2014 yılları arasında 14.587 KL olgusu bildirilmiştir. Bu olgular arasında kesin KKL tanısı alanlar tam olarak bilinmemekle birlikte, bu durumda olan hastaların lezyonlarında parazit bulunduğu ve vektör kum sineklerine bulaşımı devam ettirdiği için hastalığın kontrolünde ve sürveyansında önemleri bulunmaktadır. Leishmaniasis tedavisi açısından da ihmal edilmiş hastalıklardan biridir. Halen tedavinin temelini oluşturan beş değerli antimon bileşikleri 1940'lı yıllarda geliştirilmiş olmasına karşın KKL hastalarının büyük bölümünde tedavide güçlükler yaşanmaktadır. Bu çalışmada, oral yol ile kullanılan bir alkilfosfolipid analoğu olan miltefosin'in, standart tedavide kullanılan beş değerli antimon bileşiklerine göre tedavi etkinliğinin in vitro olarak ve daha sonra fare modellerinde in vivo olarak karşılaştırılması amaçlanmıştır. Türkiye'den beş KKL hastasının lezyonundan alınan klinik materyallerden zenginleştirilmiş besiyerinde üretilen ve kriyoprezerve edilerek sıvı azot tankında saklanan Leishmania promastigotları canlandırılmak suretiyle, yine zenginleştirilmiş besiyerinde kültürleri yapılmış ve elde edilen promastigotlardan DNA ekstraksiyonu sonrası gerçek zamanlı ITS-1 bölgesi kullanılarak tür tayini yapılmıştır. Bu beş izolata, meglumin antimoniat (MA) ve miltefosine karşı etkinliklerinin saptanması için iki ayrı yöntem ile (hemositometre lamı ve XTT testi) in vitro direnç testleri uygulanmıştır. in vitro testlerin sonucunda, tüm izolatların miltefosin ve MA'ya karşı duyarlı olduğu saptanmıştır. Genotiplendirme sonuçları, tüm suşların L. tropica türü olduğunu ortaya koymuştur. in vivo model oluşturmak amacıyla, her birinde aynı ağırlık ve yaştaki toplam 5 erkek Mus musculus (Balb/C fare) bulunan üç grup oluşturulmuştur. Bu modelde, bir KKL hastasından alınmış ve besiyerinde üretilerek sonrasında kriyoprezervasyon yapılmış promastigotlar kullanılmıştır. İnokülasyondan sonraki 12 hafta boyunca lezyon gelişimi iki haftada bir kez, düzenli olarak kontrol edilmiştir. Enfeksiyonun oluşturulduğu tarihi takip eden 13. haftadan itibaren 3 hafta süren tedavi şeması uygulanmış ve 24. haftaya kadar takip edilen fareler 24. haftada sakrifiye edilmişlerdir. Sakrifiye edilen farelerin ayak tabanlarından yapılan sürüntü ve doku süspansiyonlarına; mikroskobi, kültür ve moleküler yöntemler uygulanmıştır. Mikroskobide, miltefosin grubunda bir örnekte amastigotlar izlenirken, MA grubunda dört pozitif, kontrol grubunun ise tamamı pozitif izlenmiştir. Kültür ve moleküler yöntemlerle, miltefosin grubunda bir pozitif, MA grubunun dördünde ve kontrol grubunun tamamında pozitif sonuç alınmıştır. Bu çalışmanın sonuçları ile, tedavisinde zorluklar yaşanan KKL enfeksiyonlarında MA'a karşı olası direnç varlığı ve bu kliniğe sahip hastalarda alternatif olarak miltefosin kullanımının olası yararları hakkında değerli veriler elde edilmiştir. Ayrıca uzun süreli tedavilerde, intra-lezyoner veya parenteral verilen invaziv MA uygulamaları yerine, oral alınan miltefosinin, hasta uyumluluğunu arttırarak bu vakaların tedavisinde olumlu sonuçlar doğuracağı öngörülmektedir.
Leishmaniasis is a vector-borne protozoal disease of the tropical and subtropical regions of the world that is listed in the list of neglected diseases by the World Health Organization (WHO) and causes infections with different clinical presentations. There are three basic clinical forms of leishmaniasis that cause infection in millions of people around the world; visceral leishmaniasis (VL) characterized by involvement of internal organs, cutaneous leishmaniasis (CL) causing lesions with skin involvement, and muco-cutaneous leishmaniasis (MCL) affecting mucosa and skin. CL, usually with one or more long-standing cutaneous lesions, without causing fever or general symptoms, can be divided into two major groups; acute CL (ACL) and chronic CL (CCL). If the lesions of CL persists for 2 years or if they do not heal spontaneously, this condition is called CCL. Reporting of CL is mandatory in Turkey since the 1980s. According to the data of the Ministry of Health, 14,587 CL cases have been reported between years 2005-2014. Among these cases, definite number of CCL patients is not fully known but the parasites persist in the lesions of these patients which enables the vector sand flies to get infected and transmit the disease so they are important in the control and the surveillance of the disease. Leishmaniasis is also one of the neglected diseases in terms of treatment. Despite the fact that the pentavalent antimonial compounds which are currently the basis of treatment have been developed in the 1940's, treatment difficulties are present in most of the CCL patients. In this study, it was aimed to compare in vitro and in vivo (using mouse models) treatment efficacy of miltefosin, an orally used alkylphospholipid analogue, with the pentavalent antimonial compounds used in standard therapy. Cryopreserved Leishmania promastigotes produced in enriched medium from clinical materials taken from the lesions of five CCL patients from Turkey were thawed and cultured in enriched media and genotyping was performed using real-time ITS-1 region following DNA extraction from the resulting promastigotes. in vitro resistance tests were performed using two separate methods (hemasitometer slip and XTT test) in order to detect the activities of meglumine antimoniate (MA) and miltefosine against these five isolates. The results of the in vitro tests showed that all the strains were sensitive against miltefosine and MA. The genotyping of the strains showed that all the strains were L. tropica. In order to maintain an in vivo model, three groups were formed, each consisting of 5 male Mus musculus (Balb / C mice) of the same weight and age. In this model, cryopreserved promastigotes which were obtained from a CCL patient were used. During the 12 weeks following inoculation, lesion development was regularly checked in every two weeks. From the 13th week following the date on which the infection was established, a 3 week treatment schedule was administered and mice followed up to 24 weeks were sacrificed at 24th week. Microscopy, culture and molecular methods were applied to the swabs and tissue suspensions obtained from the foot pads and tissue suspensions of the sacrificed mice. In microscopy, one of the miltefosine group was positive, four were positive in the MA group and all were positive in the control group. By culture and molecular methods, a sample was positive in the miltefosine group, four were positive in the MA group and all samples were positive in the control group. The results of this study have provided valuable data regarding the possible presence of MA resistance in CCL infections that are difficult to treat and the possible benefits of miltefosine as a treatment alternative for patients with this clinical presentation. It is also predicted that oral treatment with miltefosine will increase the patient compliance and lead to positive outcomes in long-term treatments of these cases, rather than invasive MA therapy which is applied intra-lesionally or parenterally.
Leishmaniasis is a vector-borne protozoal disease of the tropical and subtropical regions of the world that is listed in the list of neglected diseases by the World Health Organization (WHO) and causes infections with different clinical presentations. There are three basic clinical forms of leishmaniasis that cause infection in millions of people around the world; visceral leishmaniasis (VL) characterized by involvement of internal organs, cutaneous leishmaniasis (CL) causing lesions with skin involvement, and muco-cutaneous leishmaniasis (MCL) affecting mucosa and skin. CL, usually with one or more long-standing cutaneous lesions, without causing fever or general symptoms, can be divided into two major groups; acute CL (ACL) and chronic CL (CCL). If the lesions of CL persists for 2 years or if they do not heal spontaneously, this condition is called CCL. Reporting of CL is mandatory in Turkey since the 1980s. According to the data of the Ministry of Health, 14,587 CL cases have been reported between years 2005-2014. Among these cases, definite number of CCL patients is not fully known but the parasites persist in the lesions of these patients which enables the vector sand flies to get infected and transmit the disease so they are important in the control and the surveillance of the disease. Leishmaniasis is also one of the neglected diseases in terms of treatment. Despite the fact that the pentavalent antimonial compounds which are currently the basis of treatment have been developed in the 1940's, treatment difficulties are present in most of the CCL patients. In this study, it was aimed to compare in vitro and in vivo (using mouse models) treatment efficacy of miltefosin, an orally used alkylphospholipid analogue, with the pentavalent antimonial compounds used in standard therapy. Cryopreserved Leishmania promastigotes produced in enriched medium from clinical materials taken from the lesions of five CCL patients from Turkey were thawed and cultured in enriched media and genotyping was performed using real-time ITS-1 region following DNA extraction from the resulting promastigotes. in vitro resistance tests were performed using two separate methods (hemasitometer slip and XTT test) in order to detect the activities of meglumine antimoniate (MA) and miltefosine against these five isolates. The results of the in vitro tests showed that all the strains were sensitive against miltefosine and MA. The genotyping of the strains showed that all the strains were L. tropica. In order to maintain an in vivo model, three groups were formed, each consisting of 5 male Mus musculus (Balb / C mice) of the same weight and age. In this model, cryopreserved promastigotes which were obtained from a CCL patient were used. During the 12 weeks following inoculation, lesion development was regularly checked in every two weeks. From the 13th week following the date on which the infection was established, a 3 week treatment schedule was administered and mice followed up to 24 weeks were sacrificed at 24th week. Microscopy, culture and molecular methods were applied to the swabs and tissue suspensions obtained from the foot pads and tissue suspensions of the sacrificed mice. In microscopy, one of the miltefosine group was positive, four were positive in the MA group and all were positive in the control group. By culture and molecular methods, a sample was positive in the miltefosine group, four were positive in the MA group and all samples were positive in the control group. The results of this study have provided valuable data regarding the possible presence of MA resistance in CCL infections that are difficult to treat and the possible benefits of miltefosine as a treatment alternative for patients with this clinical presentation. It is also predicted that oral treatment with miltefosine will increase the patient compliance and lead to positive outcomes in long-term treatments of these cases, rather than invasive MA therapy which is applied intra-lesionally or parenterally.
Açıklama
Anahtar Kelimeler
Leishmaniasis, Kronik kutanöz leishmaniasis, Tedavi, Miltefosin, Meglumin Antimoniat, In Vivo, Chronic Cutaneous Leishmaniasis, Treatment, Miltefosine, Meglumine Antimoniate
