Validacija nove mikroskopske metode u dijagnostici bakterijskih vaginoza primenom PCR i real-time PCR

Abstract

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 specimens 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.