Validacija nove mikroskopske metode u dijagnostici bakterijskih vaginoza primenom PCR i real-time PCR
Validation of novel microscopic method in diagnosis ob bacterial vaginosis by PCR and realtime PCR
Докторанд
Matić, SnežanaМентор
Živanović, AleksandarЧланови комисије
Ćupić, MajaDimitrijević, Aleksandra
Ljujić, Biljana
Метаподаци
Приказ свих података о дисертацијиСажетак
Uvod: Bakterijska vaginoza se smatra jednim od najčešćih poremećaja donjeg genitalnog
trakta kod žena u reproduktivnom periodu. Karakteriše je „zamena“ dominantnih vrsta
laktobacila mešanom florom anaerobnih i fakultativno anaerobnih bakterija. Pravi
uzrok bakterijske vaginoze još uvek nije utvrđen, već se dijagnoza postavlja na osnovu
kliničkih i mikroskopskih kriterijuma koji ne uzimaju u obzir specifičnu bakteriju,
već prisustvo kliničkih znakova i prisustvo bakterija određenog morfotipa na
preparatima vaginalnog razmaza.
Cilj: Ispitivanje slaganja nove modifikovane metode mikroskopiranja sa ostalim
kriterijumima (kliničkim i mikroskopskim), koji se već koriste u dijagnostici
bakterijskih vaginoza, kao i ispitivanje međusobnog slaganja svih kriterijuma sa
rezultatima molekularne analize prisustva i količine bakterija najčešće udruženih sa
„zdravim“ vaginalnim mikrobiomom (Lactobacillus spp) i bakterijskom vaginozom
(Gardnerella vaginalis i Atopobium vaginae).
Materijal ...i metode: Studija je obuhvatila 120 trudnica, starosti od 18-40 godina.
Uzorci vaginalnih briseva su uzimani tokom redovnih kontrola trudnica kada su
klasifikovani na osnovu kliničkih i mikroskopskih kriterijuma po Amselu, Nadžentu,
Ison/Haju, Klejsu i novoj modifikovanoj metodi mikroskopiranja. Zatim je sa preparata
bojenih po Gramu izolovana DNK bakterija i određivano prisustvo i količina
Lactobacillus spp, Gardnerella vaginalis, Atopobium vaginae i ukupnog vaginalnog
mikrobioma metodama PCR i real-time PCR.
Rezultati: Ustanovili smo da postoji međusobno slaganje mikroskopskih metoda, a
kliničke, mikroskopske metode i nova metoda mikroskopiranja su se slagale kada su
intermedijerni nalazi interpretirani kao normalni. Rezultati PCR-a se nisu slagali sa
rezultatima klasifikovanim po kliničkim i mikroskopskim kriterijumima. Sa druge
strane, u uzorcima preparata vaginalnog sekreta bojenih po Gramu PCR-om su
detektovani Lactobacillus spp, Gardnerella vaginalis i Atopobium vaginae, uz dominaciju
Lactobacillus spr u normalnim nalazima, a Gardnerella vaginalis i Atopobium vaginae u
nalazima bakterijske vaginoze, pri čemu je njihov odnos bio uravnotežen u
intermedijernim nalazima. Ukupni vaginalni mikrobiom je korelirao sa rezultatima
kvantitativne kategorizacije (prazan, umeren, pun) vaginalnih razmaza bojenih po Gramu,
klasifikovanih prema novoj modifikovanoj metodi mikroskopiranja, dok je relativni
odnos Lactobacillus spp i svih ostalih bakterija opadao krećući se od „normalan pun“ ka
„bakterijska vaginoza pun“, a trend relativnog odnosa G. vaginalis i A. vaginae obrnuto, je
rastao od „normalan pun“ ka „bakterijska vaginoza pun“.
Zaključak: Nova modifikovana metoda mikroskopiranja je pokazala slaganje sa
kliničkim i ostalim mikroskopskim metodama kao i kvantitativnim rezultatima
molekularne analize.
Introduction: Bacterial vaginosis is considered to be one of the most common disorders of
lower genital tract in women in reproductive age. It has been characterized by “replacement” of
dominant lactobacilli with anaerobs or facultative anaerobs. The real cause of bacterial vaginosis
still remains unclear; nevertheless its diagnosis is based on presence of clinical signs and/or
certain bacterial morhotypes in smears of vaginal swabs.
Aim: Assessment of agreement between novel modified microscopic method and other criteria
(clinical and microscopic) already in use in diagnosis of bacterial vaginosis, as well as evaluation
of agreement of aforementioned diagnostic criteria and results of molecular detection and
quantification of most common bacteria related to normal results (Lactobacillus spp) and
bacterial vaginosis (Gardnerella vaginalis and Atopobium vaginae).
Material and methods: the study enrolled 120 pregnant women between 18 and 40 years of age.
The vaginal swab speci...mens were taken during regular examinations, when they were classified
according to clinical and microscopic criteria: Amsel, Nugent, Ison/Hay, Claeys and novel
modified microscopic method. Further, bacterial DNA was isolated from Gram stained vaginal
smears preparations and detection and quantification of Lactobacillus spp, Gardnerella
vaginalis, Atopobium vaginae and total vaginal microbiome were performed by PCR and realtime PCR.
Results: Mutual agreement between all microscopic methods has been found as well as
agreement between clinical, microscopic methods and novel modified microscopic method when
intermediary results interpreted as normal. There was no agreement between PCR results and
results classified by clinical or other microscopic methods. In the other hand, Lactobacillus spp,
Gardnerella vaginalis and Atopobium vaginae in Gram stained vaginal smears were detected by
PCR, with domination of Lactobacillus spр in normal results, Gardnerella vaginalis and
Atopobium vaginae in bacterial vaginosis, and balanced presence of both bacteria in intermediary
results. Total vaginal microbiome correlated with results of quantitative categorization of Gram
stained vaginal smears according to novel modified microscopic method (empty, mid, full),
while for relative ratio of Lactobacillus spp and all other bacteria, going from “normal full”
towards “bacterial vaginosis full”, decreasing trend has been observed. Inversely, the relative
ratio of G. vaginalis and A. vaginae, going from “normal full” towards “bacterial full” had
increasing trend.
Conclusion: Agreement between novel modified microscopic method, clinical criteria and other
microscopic methods has been observed as well as agreement between novel modified
microscopic method and results of quantitative molecular analysis.
Факултет:
Универзитет у Крагујевцу, Факултет медицинских наукаДатум одбране:
25-12-2018Пројекти:
- Преклиничка испитивања биоактивних супстанци (RS-41010)