| Abstract | Cilj ovog rada je istraživanje vrijednosti PCR-RFLP metode na dijelu genoma E1 za genotipizaciju HPV, određivanje osjetljivosti rutinske probirne metode HC 2 u našim uzorcima uzetih od žena s različitim citološkim nalazom i asimptomatskih muškaraca, te na temelju dobivenih rezultata pokušati stvoriti polazne pretpostavke za uspostavu optimalnog algoritma za tipizaciju genotipova HPV u Hrvatskoj. U istraživanje je uključeno 120 ispitanika koji su podijeljeni u 4 skupine: skupina od 25 žena s CIN 1, skupina od 25 žena s CIN 2 i/ili 3, skupina od 50 asimptomatskih muškaraca koji su bili upućeni na testiranje jer njihova partnerica ima dokazanu infekciju s HPV visokog rizika, te kontrolna skupina od 20 žena s urednim citološkim nalazom. Uzorci za ispitivanje ženskih ispitanika su obrisci vrata maternice. Uzimani su prema indikacijama pri rutinskom ginekološkom pregledu. Uzorci za ispitivanje muških ispitanika su bioptat kože i/ili sluznice genitalne regije. Uzeti su ekskohleacijski uz prethodnu primjenu anestetičke kreme EMLAR (Astra-Zeneca). U skupini od 50 asimptomatskih muškaraca osjetljivost PCR metode s parom začetnika My09/11 bila je 94% (47/50), a s parom začetnika p1/p2 86% (43/50). Paralelno su uzorci testiralni HC 2 metodom koristeći probe za detekciju genotipova i visokog i niskog rizika. Osjetljivost ove metode bila je 90% (45/50). Razlika u osjetljivosti između pojedinih metoda nije se pokazala statistički značajnom (χ-kvadrat test p=0.1824; Fisherov egzaktni test p= 0.7150). Genotipizacijom je u ovoj skupini nađeno čak 9 različitih genotipova. Najčešće zastupljeni bili su HPV16 (31%), HPV6 (31%) i HPV31 (10.3%). Infekcija s HPV visokog rizika nađena je u 55.2% asimptomatskih muškaraca, što se nije pokazalo statistički značajnim u odnosu na žene s urednim citološkim nalazom (47%) (χ-kvadrat test p=0.7076). Ukupno je genotipizirano 67 žena s različitim citološkim nalazom. Žene s CIN 1 i žene s urednim citološkim nalazom imale su sličnu učestalost HPV 16 (23.5%, odnosno 28%), dok se žene s CIN 2 i/ili 3 statistički značajno razlikuju u učestalosti HPV 16 (60%) u odnosu na žene s urednim citološkim nalazom (χ-kvadrat test p=0.0198). Usporedbom genotipova prema onkogenom potencijalu u pojedinim skupinama ispitanika, statistički značajno se razlikovala samo skupina žena s CIN 2 i/ili 3 u odnosu na kontrolnu skupinu (Fisherov egzaktni test p=2.060E-04). U 96 uzoraka nađeno je 18 različitih genotipova. Od toga 8 genotipova nije uključeno u HC 2 test (HPV 73, HPV 53, HPV 66, HPV 40, HPV 54, HPV 70, HPV 61 i HPV 62). Genotipovi niskog rizika (HPV 61 i HPV 62) nađeni su probom za detekciju genotipova niskog rizika, a genotip visokog rizika (HPV 73) i genotipovi vjerojatno visokog rizika (HPV 53 i HPV 66) nađeni su probom za detekciju genotipova visokog rizika, što povećava osjetljivost testa, a ne smanjuje specifičnost (7.3% uzoraka). Problem je u uzorcima u kojima su genotipovi niskog rizika (HPV 6, HPV 40, HPV 54, HPV 70) nađeni probom za detekciju genotipova visokog rizika, ili su davali pozitivni signal s obje probe. Ovi lažno pozitivni rezultat dobiveni su u 6.3% uzoraka. Korištenjem začetnika p1/p2 umnožen je dio genoma E1 veličine 526 – 594 bp. Od 120 pozitivnih uzoraka, ovom metodom je bilo pozitivno 111; osjetljivost metode je 92.5%. S p1/p2 PCR dobivena su 3 pozitivna uzorka koja su bila negativna PCR metodom umnoženom parom začetnika MY09/MY11; tek kombinacijom metoda dobiveno je 120 pozitivnih uzoraka. Dobiveni amplifikati cijepani su s 12 restrikcijskih enzima. PCR- RFLP metoda s parom začetnika p1/p2 pokazala se slabo tipabilnom i slabo diskriminatornom u usporedbi s najčešće korištenom RFLP metodom koju su uveli Bernard i sur., a koja je korištena kao standardna metoda. Kod većine genotipova dobiven je vrlo sličan raspored i vrlo mali broj fragmenata na osnovu kojeg se nisu mogli međusobno razlikovati. Ukupno je ovom metodom tipizirano 7 genotipova (od 18 koliko je dobiveno RFLP/MY). . Kombinacijom ovih dviju metoda (RFLP/MY i RFLP/p1p2) nađene su varijacije unutar poznatih genotipova. Dobivena su 2 različita rasporeda fragmenata za HPV 16 i HPV 6. Usporedbom ovih varijanti s različitim skupinama ispitanika, nisu primijećene razlike u učestalosti pojedine varijante. |
| Abstract (english) | The aim of this study was to determine the value of the PCR-RFLP method performed on part of the gene E1 for genotyping of the HPVs; to determine the sensitivity of the HC 2 method in our samples and on the basis of our results to try to create optimal algoritham for HPV genotyping in Croatia.. In the study were included 120 men and women divided in 4 groups: a group consisted of 25 women with CIN I, a group consisted of 25 women with CIN II and/or III, a group consisted of 50 asymptomatic men; in control group there were 20 women with negative cytology. Brush swabs were used to obtain the endocervical samples from women; samples were taken according to indications during routine gynecologic examination. Bioptic samples of the skin and/or mucous membrane of the genital region were taken from men included in the study after the area was anesthetized with EMLAR cream (Astra-Zeneca). In the group of asymptomatic men the sensitivity of the PCR/MY09/11 method was 94% (47/50), of the PCR/p1p2 86% (43/50) and of HC 2 90% (45/50). The differences among these methods were not statistically significant (χ2- test p=0.1824; Fisher's test p= 0.7150). In this group was found 9 different genotypes. The most frequent genotypes were: HPV 16 (31%), HPV 6 (31%) and HPV 31 (10.3%). The frequency of infection with high risk genotypes (55.2%) was not significantly different from control group of women (47%) (χ2- test p=0.7076). Totally it was genotyped 67 samples taken from women with and without CIN (control group). It was found that the frequency of HPV 16 was similar in normal women (23.5%) and women with CIN 1 (28%), but it was found statistically significant difference in women with CIN 2 and/or 3 (60%) (χ2-test p=0.0198). Comparing genotypes according to oncogenic potential, it was found statistically significant difference only in women with CIN 2 and/or 3 (Fisher test p=2.060E-04). In 96 samples was found 18 different genotypes; 8 genotypes were not included in HC 2 test: (HPV 73, HPV 53, HPV 66, HPV 40, HPV 54, HPV 70, HPV 61 and HPV 62). Low risk genotypes (HPV 61 and HPV 62) were found with probe for detection low risk genotypes; high risk and probable high risk genotypes ( HPV 73, HPV 53 and HPV 66) were found with probe for detection high risk genotypes; it increased the sensitivity of the test, but not reduced specificity (7.3%). The problem were samples in which low risk genotypes (HPV 6, HPV 40, HPV 54, HPV 70) were found with probe for detection high risk genotypes or the signal was positive with both probes. These false positive results were found in 6.3% of samples. Using the set of general primers p1/p2 it was amplified a 526–594 bp fragment spanning the E1 open reading frame (ORF). Out of 120 samples, with this method it was positive 111; the sensitivity of the method was 92.5%. With p1/p2- PCR it was found 3 positive samples which were negative with MY09/MY11- PCR; only combining the methods were found all positive samples. . Amplified sequences were analysed by restriction endonuclease digestion. RFLP method performed on E1 was found weakly discrimintory comparing with the most used RFLP method. With this method it was totally genotyped 7 genotypes (out of 18 which were genotyped with RFLP/MY). Combining two RFLP methods we found two variants of HPV 16 and HPV 6. However, comparing these variants with different groups of women and with group of asymptomatic men, differencies were not found in frequences of the particular variants. |
