Vpeljava modernega diagnostičnega pristopa za dokaz babezij v kliničnih in živalskih vzorcih

Caf, Nika (author), Avšič Županc, Tatjana (mentor), Korva, Miša (comentor)

Babezija je parazit, ki se s klopom prenese na ljudi in živali, kjer lahko povzroči okužbo. Zlati standard dokazovanja babezioze je neposredni mikroskopski pregled barvanega krvnega razmaza, s katerimi ne moremo določiti babezije do vrste natančno. Babeziozo lahko dokažemo tudi z metodo posredne imunofluorescence, ki pa ni primerna pri imunsko oslabljenih bolnikih. Z magistrsko nalogo smo razvili PCR v realnem času za dokaz DNA babezij, ki povzročajo okužbe pri ljudeh in živalih v Sloveniji in s tem olajšali ter skrajšali čas do laboratorijskega izvida. Dokazali smo, da je metoda PCR v realnem času bolj občutljiva in bolj specifična od neposrednega mikroskopskega pregleda preparata krvi ter klasičnega PCR. Kot selekcijski marker smo uporabili gen 18S rRNA, ki vsebuje hipervariabilne odseke, obdane z visoko ohranjenimi regijami. Razvili smo visoko specifičen in občutljiv PCR v realnem času, ki pomnožuje sev humane babezije Babesia sp. Irk, ki so ga dokazali leta 2014 pri imunsko oslabljeni bolnici iz Prekmurja. S kvantifikacijo z digitalnim PCR smo spremljali potek parazitemije in zdravljenja pri bolnici ter dokazali babezijsko DNA tudi po 89. dneh zdravljenja, ko so bili rezultati ostalih metod že negativni. Zaključimo lahko, da je PCR v realnem času hitra, visoko občutljiva in enostavna metoda, ki omogoča dokaz babezioze v zgodnjih fazah bolezni ter spremljanje poteka zdravljenja.

• Language: Slovenian
• Keywords: babezioza, prenašalci, gostitelji, klopi, parazitologija, babezije, mikrobiološke metode, PCR, PCR v realnem času, digitalni PCR
• Work type: Master's thesis/paper
• Typology: 2.09 - Master's Thesis
• Organization: BF - Biotechnical Faculty
• Publisher: [N. Caf]
• Year: 2017
• PID: 20.500.12556/RUL-95707
• UDC: 616.993.19:579.67.083
• COBISS.SI-ID: 4818552
• Publication date in RUL: 21.09.2017

Secondary language

• Language: English
• Title: Introduction of modern diagnostic approach to detect Babesia in clinical and animal samples

Abstract

Babesia is a parasite that can be transmitted to humans and animals, where it causes infection. With methods like microscopic staining of blood smears, that do not give us the information about the species, and indirect immunofluorescence, that we can not use on immunocompromised patients, real time PCR seems to be a much better choice for diagnostics. Within M. Sc. Thesis we developed real time PCR for detection of Babesia species DNA, found in humans and animals around Slovenia. We proved that real time PCR is a more sensitive and specific method than the microscopic staining of blood smears and conventional PCR. As a selection marker we used 18S rRNA gene with hipervariable sections that are surrounded with highly conservative regions. Our real time PCR amplifies strain of human Babesia sp. Irk, that was first found in 2014 in an immunocompromised patient from Prekmurje. By using absolute quantification of a digital PCR, we were able to follow parasitemia and treatment of the patient and detected DNA of the parasite even after 89 days from the beginning of the treatment, when all the other methods showed negative results. In conclusion, we can say that real time PCR is a quick, highly sensitive method that is easy to use and is useful for detection of babesial DNA at the beginning of the infection as well as for monitoring the course of treatment.

• Keywords: babesiosis, vectors, hosts, ticks, parasitology, babesia, microbiology methods, PCR, real time PCR, digital PCR