Cryptosporidiosis in Humans with Reference to the First Case of Cryptosporidium hominis Infection in Turkey

Amaç: Cryptosporidiosis dünya çapında bir zoonozdur. Mikroskopik muayene, etken türlerin belirgin olmayan morfolojik özellikleri nedeniyle başarısız olabilir. Bu nedenle moleküler tanı daha da önem kazanmıştır.Yöntemler: Cryptosporidium spp. oosistlerini belirlemek için 150 hastanın dışkı örnekleri karbol-fuksin boya kullanılarak incelendi. Pozitif örneklerde farklı türlerin belirlenmesi için nested polimeraz zincir reaksiyonu-restriksiyon parça uzunluk polimorfizmi (PZR-RPUP) kullanıldı. Örnekler, diğer parazitler için yaş preparasyon ve çinko sülfat flotasyonu yöntemleriyle de araştırıldı.Bulgular: Mikroskobik muayene ve moleküler yöntemlerle sırasıyla %0,67 (1/150) ve %8,93 (5/56) pozitiflik saptandı. Nested PZR-RPUP bir örnekte Cryptosporidium hominis'in (C. hominis), dört örnekte ise Cryptosporidium parvum'un (C. parvum) saptanmasına olanak verdi. Bu çalışma ile C. hominis Türkiye'de ilk kez insanlarda bildirildi. Enfekte olanlar arasında üçü çocuktu, dışkısında C. parvum oosistleri görülen dört hastada gastroenterit, C. hominis pozitif olan bir hastada ise mide bulantısı ve kusmanın eşlik ettiği gastroenterit vardı. Enfekte kişilerin hiçbirinde Giardia spp. ve Entamoeba spp. saptanmadı.Sonuç:­ C. parvum olgularının C. hominis olgularından fazla olması, enfekte kişilerin hayvancılık yapılmasına izin verilmeyen kentsel alanda yaşamasına rağmen, bir zoonotik bulaşma olduğunu düşündürmektedir. Bununla birlikte, şehir suyundaki su kaynaklı patojen kirliliği bir bulaşma faktörü olarak düşünülmektedir.Aim: Cryptosporidiosis is a worldwide zoonosis. Microscopic examinations may fail due to indistinctive morphological peculiarities of causative species. Hence, molecular diagnostics has become more important. Methods: Stool samples from 150 patients were examined using carbol-fuchsin stain to determine Cryptosporidium spp. oocysts. Combined nested polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) technique was used for establishing different species in positive samples. The samples were also screened for other parasites by wet-mount and zinc sulfate flotation methods. Results: Microscopic examinations and molecular techniques revealed 0.67% (1/150) and 8.93% (5/56) positivity, respectively. Nested PCRRFLP enabled the detection of Cryptosporidium hominis (C. hominis) in one sample, while Cryptosporidium parvum (C. parvum) was detected in four samples. With this study, C. hominis was reported from humans for the first time in Turkey. Among infected ones, three of which were children, four patients excreted C. parvum oocysts had gastroenteritis, and a patient positive for C. hominis had gastroenteritis accompanied by nausea and vomiting. No Giardia spp. and Entamoeba spp. were detected in all infected individuals. Conclusion: C. parvum cases outnumbered C. hominis cases, suggesting a zoonotic transmission although infected individuals were living in an urban area where animal husbandry was not allowed. However, water-borne pathogen contamination in the city's water supply is considered a factor for transmission

Investigation of Giardia spp. existence by using microscopic method and PCR in human in Tekirdag

Giardiosis, tüm dünyada ve Türkiye’de, insanlarda ve hayvanlarda görülen zoonotik bir protozoon hastalığıdır. Hastalığın klinik karakteri subklinikten, şiddetli ishal ile karakterize akut forma kadar değişir. Etkeni Giardia cinsinde yer alan kamçılı protozoonlardır. Başlıca bağırsaklarda etkili olan parazitin doğadaki döngüsü temel olarak fekal oral bulaşma esasına dayanır. Dışkı ile atılan kist formları, değişen çevresel koşullara oldukça dirençli olduğundan ve özellikle sulu ortamda uzun süre canlı kalabildiğinden, hastalığa genelde yaygın olarak rastlanır. Bu tezde, 01.01.2011- 31.12.2011 tarihleri arasında Tekirdağ Devlet Hastanesine özellikle sindirim sistemi şikayeti ile başvuran 573 hastadan gaita numuneleri alınmış ve söz konusu süreçte hastaya ve gaita numunelerine yönelik temel parametreler kaydedilmiştir. Takibinde numuneler natif muayene ve doymuş çinko sülfat flotasyon tekniği kullanılarak, mikroskop altında Giardia spp. kistleri yönünden incelenmiştir. Bu numunelerden, kist yönünden pozitif olanlar ve hastalığın subklinik formuna sahip olabileceği düşünülen örneklerden 90’ı seçilmiş ve DNA ekstraksiyonu gerçekleştirilmiştir. Takibinde Giardia duodenalis spesifik, ß-giardin gen bölgesine yönelik primerler kullanılarak nested PCR yapılmış ve ürünler HaeIII enzimi ile kesilerek poliakrilamid jel elektroforez-PAGE’de yürütülmüştür. Sonuç olarak, natif muayenede 21 (%3,66), doymuş çinko sülfat flotasyonu ile 26 (%4,54), nested PCR ile 27 (%4,71) pozitif elde edilmiştir. Sınırlayıcı enzim parça uzunluk çeşitliliği-RFLP verilerine göre, pozitif örneklerin 15’inin (%55,56) assemblage A, 2’sinin (%7,41) assemblage B ve 10’unun (%37,03) assemblage B ve assemblage E miks olduğu anlaşılmıştır. Kaydedilen verilerle sonuçlar karşılaştırıldığında, Tekirdağ’da insan giardiosisinin belli bir önem taşıdığı ve hastalığın zoonotik dinamiğinin bölgede önemli bir role sahip olabileceği görülmüştür. Yine bu tez çalışmasıyla, dünyada ilk defa insanlarda bu derecede yoğun assemlage E bildirimi yapılmakta olup, ilgili olguların belirgin klinik öneme sahip olması, söz konusu türe yönelik daha ayrıntılı çalışmalar yapılması gerektiğini göstermiştir.Giardiosis is a zoonotic protozoan disease, which is seen worldwide, including in Turkey. Clinical feature of the disease varies from subclinical infection to overt, acute form characterised with severe diarrhea. The agent is flagellated protozoan in Giardia genus. Natural life cycle of the parasite, which especially affects the small intestine, is based on primarily faecal-oral transmission route. The disease is prevalent because the cyst form, excreated with faeces, is highly resistant to different environmental conditions, and can survive for a long period especially in water. In this study, single stool samples were collected from the 573 patients who applied to the public hospital especially with complaints of gastrointestinal symptoms, between 01 January 2011-31 December 2011, in Tekirdag, and in this period, all the information were recorded, about the faeces specimen and the patients. Subsequently, the samples were examined using wet-mount and saturated zinc sulfate centrifugal flotation method, and the slides were screened for the cysts of Giardia spp. under light microscope. Of the total specimen, 90 samples, which were either cyst positive or negative but obtained from the patients who were suspected of having subclinical form of the disease, were selected and DNA extraction was performed. After that, nested PCR was conducted using Giardia duodenalis specific primers targeted to DNA sequences of ß-giardin gene region, and the yields of the PCR were restricted with endonuclease HaeIII and run on polyacrylamide gel electrophoresis-PAGE. As a result, 21 (3.66%), 26 (4.54%) and 27 (4.71%) positive were detected in the wetmount, saturated zinc sulfate centrifugal flotation, and nested PCR methods respectively. According to the restriction fragment length polymorphism-RFLP, it was observed that out of the positive samples, 15 (55.56%) were assemblage A, 2 were (7.41%) assemblage B, and 10 (37.03%) were mix of assemblage B and assemblage E. It was evaluated that human giardiosis has a certain importance in Tekirdag, and zoonotic cycle of the disease should have an important role in the region. In this thesis, assemblage E infection, at this high prevalence level in humans, is reported first here, and serious clinical features of the related cases pointed to the need for more detailed studies on this assemblage

Cryptosporidiosis in Humans with Reference to the First Case of Cryptosporidium hominis Infection in Turkey

Aim: Cryptosporidiosis is a worldwide zoonosis. Microscopic examinations may fail due to indistinctive morphological peculiarities of causative species. Hence, molecular diagnostics has become more important. Methods: Stool samples from 150 patients were examined using carbol-fuchsin stain to determine Cryptosporidium spp. oocysts. Combined nested polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) technique was used for establishing different species in positive samples. The samples were also screened for other parasites by wet-mount and zinc sulfate flotation methods. Results: Microscopic examinations and molecular techniques revealed 0.67% (1/150) and 8.93% (5/56) positivity, respectively. Nested PCR-RFLP enabled the detection of Cryptosporidium hominis (C. hominis) in one sample, while Cryptosporidium parvum (C. parvum) was detected in four samples. With this study, C. hominis was reported from humans for the first time in Turkey. Among infected ones, three of which were children, four patients excreted C. parvum oocysts had gastroenteritis, and a patient positive for C. hominis had gastroenteritis accompanied by nausea and vomiting. No Giardia spp. and Entamoeba spp. were detected in all infected individuals. Conclusion: C. parvum cases outnumbered C. hominis cases, suggesting a zoonotic transmission although infected individuals were living in an urban area where animal husbandry was not allowed. However, water-borne pathogen contamination in the city’s water supply is considered a factor for transmission

Cryptosporidium parvum oocystlerindeki zamana ba?lı canlılık de?işim derecesi vital boyalarla belirlenebilir mi?]

The present study was undertaken to determine time dependent viability changes of purified Cryptosporidium parvum oocysts, stored in antimicrobial-supplemented PBS at +4 degrees C, using vital dyes (DAPI/PI). The trials demonstrated that vital dyes could provide estimation of oocyst viability, and furthermore, if interpreted correctly, they could be used to determine the degree of the viability in Cryptosporidium parvum oocysts as cell culture-PCR assay is used