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ID naloge: 118 Letnik: 2002 Predmet: mikrobiologija in imunologija
Neposredno dokazovanje in genotipizacija povzrocitelja bolezni macje opraskanine
Avtor: Katja Triller, Petra Štrus
Mentor: prof. dr. Tatjana Avšic-Županc
Somentor: dr. Miroslav Petrovec
IZHODIŠCE. Bolezen macje opraskanine je eden izmed vzrokov klinicno izražene limfadenopatije. Zgodnje prepoznavanje bolezni je za diagnosticno in nadaljnjo terapevtsko obravnavo bolnika velikega pomena. Omenjena bolezen je zoonoza. Pomemben rezervoar so macke, s katerimi prihajajo v stik zlasti otroci, ki zato najpogosteje zbolijo. Glede na študije v Evropi in ZDA je povzrocitelj bolezni bakterija Bartonella henselae. Klasicne metode diagnostike so težavne in dolgotrajne, zato so se uveljavile novejše, zlasti molekularne diagnosticne metode.
NAMEN. Študij, ki bi omogocale do vrste in genotipa natancno opredelitev povzrocitelja bolezni macje opraskanine, v Sloveniji še ni bilo. Z raziskavo smo želeli ugotoviti, katere vrste ali genotipi bartonel so prisotni pri bolnikih s klinicnimi znaki bolezni. Hkrati smo želeli oceniti uporabnost pomnoževanja dveh odsekov bakterijskega genoma (dela gena htrA ter medgenskega predela ITS) za med- in znotraj- vrstno razlikovanje bakterij v rodu Bartonella.
HIPOTEZA. Predvidevali smo, da bomo iz vzorcev bezgavk bolnikov s klinicno sliko bolezni macje opraskanine osamili bakterijo vrste Bartonella henselae. Pricakovali smo, da bomo, s pomnoževanjem in analizo razlicnih odsekov genoma, dolocili genotipe B. henselae, ki so prisotni v slovenskem prostoru.
METODE. V raziskavo smo vkljucili 24 nakljucno izbranih vzorcev, odvzetih pri 21 pacientih, ki so bili vecinoma punktati ali aspirati prizadetih bezgavk, pa tudi kri in likvor. Iz vzorcev smo najprej osamili DNK. Z verižno reakcijo s polimerazo smo pomnožili odseka gena htrA in medgenskega predela ITS ter nato z analizo nukleotidnega zaporedja pomnoženih odsekov ter primerjavo z zaporedji v genski banki dolocili genotip prisotnega mikroorganizma.
REZULTATI. Pri vzorcih 11 razlicnih bolnikov smo uspešno pomnožili del gena htrA. Z analizo nukleotidnega zaporedja omenjenega dela genoma smo po primerjavi s podatki v genski banki ugotovili, da pomnožena DNK pripada bakteriji vrste Bartonella (Rochalimaea) henselae. Analizirana zaporedja so bila pri vseh preucevanih vzorcih popolnoma enaka. V vzorcih 10 razlicnih bolnikov smo pomnožili del medgenskega predela ITS. Nukleotidno zaporedje pomnoženih odsekov se je med vzorci razlikovalo na 3 znacilnih mestih. Po primerjavi z zaporedji v genski banki smo dokazali 2 razlicna genotipa (Houston-1 in CAL1), ki pripadata locenima genskima skupinama (Houston-1 in Marseille) bakterije vrste Bartonella henselae.
ZAKLJUCKI. Na osnovi naše raziskave smo potrdili hipotezo, da je v Sloveniji v vecini primerov povzrocitelj bolezni macje opraskanine Bartonella henselae, ki se pojavlja v dveh že opisanih genotipskih razlicicah. Za medvrstno razlikovanje povzrocitelja je primerno pomnoževanje in sekvencna analiza odseka medgenskega predela ITS.
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[Abstract / English version]
Neposredno dokazovanje in genotipizacija povzrocitelja bolezni macje opraskanine
Author: Katja Triller, Petra Štrus
Mentor: prof. dr. Tatjana Avšic-Županc
Co-mentor: dr. Miroslav Petrovec
BACKGROUND. Cat-scratch disease is one of the causes of clinically evident lymphadenopathy. Early recognition of the disease is crucial for further diagnostic and therapeutic investigations. The disease is considered zoonosis and the cats are a big natural reservoir. Since children come in contact with cats very frequently, the cat-scratch disease is most common in this age group. The causative agent is a small bacillus, Bartonella henselae, which is considered an emerging pathogen. In the case of Bartonella, the classical diagnostic methods are time-consuming and unreliable. To establish a precise diagnosis of infection (to the species or possibly genotype level), newer, molecular methods of diagnosis are applied.
AIM. There have not been any studies in Slovenia yet, which could confirm the presence of particular species or genotypes of Bartonella in patients with cat-scratch disease. The aim of the present study was to find out, which bacterial species or genotypes are present in lymph nodes of patients with clinically manifested disease. Two different parts of the genome were analysed (a part of the htrA gene and intergenic spacer region) for inter- and intra- species differentiation of genus Bartonella.
HYPOTHESIS. Based on the various studies in Europe and USA we expect to isolate species Bartonella henselae from lymph node specimens of patients with clinical signs of cat- scratch disease. With polymerase chain reaction and sequence analysis, we further expect to determine the genotypes of Bartonella henselae.
METHODS. Our study included 24 randomly chosen samples, taken from 21 different patients. The samples were mostly lymph node specimens, but also blood and cerebrospinal fluid. After DNA extraction, part of the htrA gene and ITS intergenic spacer were amplified by polymerase chain reaction. The nucleotide sequences of the amplified parts were determined and compared with those in GenBank to establish the genotype.
RESULTS. We successfully amplified the part of the htrA gene in samples from 11 different patients. The sequence analysis confirmed the presence of Bartonella henselae species in all positive samples. The intergenic spacer region was amplified in samples from 10 different patients. The analyzed sequences differed in 3 characteristic positions. By comparing them with the sequences in the GenBank, the presence of 2 different genotypes (Houston-1 and CAL1) of Bartonella henselae which belong to two separate genogroups (Houston-1 and Marseille, respectively) was confirmed.
CONCLUSIONS. We can therefore conclude that Bartonella henselae is the causative agent of cat-scratch disease in Slovenia and that there are members of both genogroups present. The part of intergenic spacer region is useful for inter- species differentiation among members of the genus Bartonella.
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