Molekularna dijagnostika urogenitalnih infekcija uzrokovanih bakterijom Chlamydia trachomatis s područja Slavonije i Baranje

U Službi za mikrobiologiju Zavoda za javno zdravstvo Osječko-baranjske županije je ukupno prikupljeno i mikrobiološki obrađeno 925 uzoraka na prisutnost bakterije Chlamydia trachomatis (CT) obrađenih in vitro COBAS TaqMan CT testom za dijagnostiku urogenitalnih infekcija na COBAS TaqMan 48 uređaju. Obrađeno je 159 (17.2%) uzorka mokraće, 458 (49.5%) obrisaka vrata maternice i 344 (37.3%) obriska mokraćovoda. Otkrivene su 23 (2.5%) urogenitalne infekcije uzrokovane CT što je dvostruko više od broja pozitivnih uzoraka (1.3 %) otkrivenih korištenjem imunoflorescentne dijagnostike. Obzirom na godišnji ulaz od 3.000 uzoraka za testiranje na CT, molekularna dijagnostika ima veliku prednost u otkrivanju asimptomatskih i simptomatskih infekcija u odnosu na imunoflorescentnu dijagnostiku

Distribution of Genital Human Papillomavirus (HPV) Genotypes in Croatian Women with Cervical Intraepithelial Neoplasia (CIN) – A Pilot Study

Genital infection with high-risk human papillomavirus (HR HPV) associates with increased risk of developing pre- cancerous lesions, such as cervical intraepithelial neoplasia (CIN). The objective of this pilot study conducted in north- -east Croatia was to determine the prevalence of HPV genital infection in women with abnormal cervical cytology and to determine its association with their age and HPV genotype(s). From March 2009 to December 2011, cervical swabs from 100 women were analysed for HR HPV infection (AMPLICOR HPV Test, Roche Diagnostics) and genotyped for high risk (HR), intermediate (IR) and low risk (LR) HPVs (Linear Array HPV Genotyping Test, Roche Diagnostics). The most prevalent HR genotypes in women with CIN were HPV 16 (27.6%), HPV 31 (11.8%), HPV 51 and HPV 52 (10.2% each). The most prevalent IR genotypes were HPV 66 (30%) and HPV 62 (23.3%). The most prevalent LR genotype was HPV 6 (20.3%). Women between 21 and 25 years of age showed the highest rate of HPV infection (44.2%). Moreover, women younger than 35 years showed a significant association (p<0.01) and positive correlation (r=0.67; p<0.05) between HR HPV infection and CIN stages 1 and 2. Multiple HPV infections were found in almost half of the women. This is the first study that analysed the prevalence of genital infection with HR/IR/LR HPVs in women with CIN from north-east Croa- tia. Despite the preliminary nature of this pilot study, the lower prevalence of some HR HPVs (HPV18) and the higher prevalence of other HR HPVs (HPVs 51, 52 and 31) may imply the necessity for the development of more targeted anti- -HPV vaccines or other strategies for more efficient protection against oncogenic HPV infection in women from our region

PRKAG3 Gene and Meat Quality of Hybrid Pigs

The study was performed on 89 PIC (Pig Improvement Company) pig carcasses with the aim to investigate influence of I199V polymorphism at PRKAG3 locus on meat quality traits. After the slaughter pH45 and pH24 in MS (semimembranosus) and LD (longissimus dorsi) muscle, CIE-L*a*b*, drip loss, cooking loss and shear force were measured and pigs were genotyped for PRKAG3 Ile199Val polymorphism. Genotype frequencies at 199th codon of the PRKAG3 gene were 80.90%, 11.24% and 7.87% for Val/Val, Ile/Val and Ile/Ile genotype, respectively. Influence of I199V polymorphism was observed for pH45 values in MS and LD muscle, b*, drip loss and shear force values, where Ile/Ile genotype showed preferable values in all investigated traits

Prevalence and Genotype Distribution of High-risk Human Papillomavirus (HR HPV) in Male Genital Samples of Osijek-Baranja County

This is a first cross-sectional study on the prevalence and distribution of HPV infection in asymptomatic, heterosex- ual men from Osijek-Baranja County, Croatia. Between 2009 and 2011, 330 men tested for sexually transmitted diseases (STDs) were recruited. Their genital swabs were tested for high-risk HPV (HR HPV) infection by the AMPLICOR HPV test and further genotyped by the Linear Array HPV Genotyping Test (both by Roche). Infection with a single HR HPV was detected in almost one third of men (39%) whereas multiple HPV types, in more than a half of HR HPV-positive men (61%). The highest HR HPV prevalence was detected in those younger than 20 (37.5%) and lowest in 31–35 year old men (27.8%). The most common genotypes were HPV 6 (24%), 16 (17.8%), 51 (9%), 52 (6%), 35, 55, 66, 84 (each 5%), 31, 62 (each 4%), 39, 58, 59, 83 (each 2.5%), and finally 56, 18, 53, and 54 (each 1.3%). Having more than one sexual partner per year was significantly associated with HR HPV infection in age group between 26 and 30 years (p=0.001). Due to the high prevalence of HR HPV infection in men of this County and its risk of transmission to women, we recommend more public awareness about this particular STD and initiating vaccination programs of young men and women

Čimbenici rizika i molekularne predispozicije za displaziju vrata maternice u žena iz istočne Hrvatske

Purpose: The aim of this study was to investigate possible association between high-risk Human papillomavirus (HR HPV) – induced cervical infection, HR HPV-related cervical dysplasia, HR HPV genotypes with two Toll-like receptor (TLR) 9 gene polymorphisms and other risk factors. Methods: During a three-year period, 100 women positive for cervical HR HPV infection (97 with cervical dysplasia and 3 positive women without dysplasia) were genotyped using the Linear Array HPV Genotyping assay (Roche Diagnostics). Furthermore, two polymorphisms of TLR9 (-1486T/C, rs187084 and 2848C/T rs352140) were determined using real-time Polymerase Chain Reaction; 50 HR HPV negative women of similar ethnicity were included as controls. Results: This study showed that infections with HPV 16 in women with cervical dysplasia were more frequently found compared with HPV 18 infections (p=0.0539). Comparison between HR HPV positive and negative women showed significant association between age >35 years (p=0.0058), being unmarried women (p=0.0001), nocondom usage (p=0.0304) and active tobacco smoking (p=0.0376) with HR HPV cervical infection. No significant associations between two TLR9 gene polymorphisms, HR HPV infection and cervical dysplasia were found. Conclusion: Our results indicated that: i) women with cervical dysplasia showed significant higher rate of HR HPV 16 infection compared to HR HPV 18, ii) HR HPV – infection was strongly correlated with social risk factors and iii) TLR9 gene polymorphisms (rs187084; rs352140) did not correlate with HR HPV infection and cervical dysplasia. Further genome-wide association studies could open new frontier in understanding the relationship between polymorphisms at TLR9 and immunological mechanisms in HPV-induced carcinogenesis.Cilj: Cilj ovog istraživanja bio je utvrditi povezanost između infekcije vrata maternice visokorizičnim humanim papilomavirusima (engl. high-risk Human papillomavirus - HR HPV), displazije vrata maternice uzrokovane visokorizičnim genotipovima HPV-a, visokorizičnih genotipova HPV-a te dva polimorfizma gena za Toll-u sličan receptor (TLR) 9 i drugih rizičnih čimbenika. Metode: Tijekom trogodišnjeg razdoblja, u 100 žena s cervikalnom infekcijom visokorizičnim genotipovima HPV-a (97 s displazijom vrata maternice i 3 s pozitivnim nalazom HPV-a ali bez displazije vrata maternice) određeni su genotipovi virusa primjenom molekularnog testa Linear Array HPV Genotyping assay (Roche Diagnostics). Također su analizirana i dva polimorfizma gena za TLR9 (-1486T/C, rs187084 i 2848C/T rs352140) koristeći metodu lančane reakcije polimerazom u stvarnom vremenu; te je 50 žena s negativnim hrHPV ili sličnog etniciteta uključeno kao kontrolna skupina. Rezultati: Istraživanje je pokazalo da su HR HPV infekcije genotipom 16 češće u žena sa displazijom vrata maternice u usporedbi sa ženama s infekcijom genotipa 18 (p=0.0539). Usporedbom žena s pozitivnim i negativnim nalazom na HR HPV, utvrđena je značajna povezanost između dobi >35 godina (p=0.0058), bračnog statusa (slobodne osobe, p=0.0001), nekorištenja kondoma (p=0.0304) i aktivnog pušenja (p=0.0376) i infekcije s HR HPV.“ Povezanost između dva polimorfizma gena za TLR9, infekcije HR HPV i displazije vrata maternice nije dokazana. Zaključak: Rezultati ovog istraživanja pokazali su da: i) je u žena sa displazijom vrata maternice značajno veća učestalost infekcije s genotipom 16 u usporedbi s genotipom 18, ii) infekcija s HR HPV povezana je sa socijalnim faktorima rizika i iii) polimorfizmi gena za TLR9 (rs187084; rs352140) nisu povezani s HR HPV infekcijom i displazijom vrata maternice. Daljnja genomska istraživanja mogu proširiti spoznaje u razumijevanju odnosa između polimorfizma gena za TLR9 i imunoloških mehanizama u HPV-om induciranoj karcinogenezi

DNA isolation from Aspergillus flavus: Optimal method selection

The methods for fungal genomic DNA isolation for PCR amplification, including commercially available kits, must often be adapted in order to produce sufficient amounts of high-quality DNA from specific fungal species. The aim of this study was to select an optimal method for the isolation of DNA from Aspergillus flavus suitable for PCR reaction. Four different methods were compared according to their efficiency in isolating pure DNA, their price and time consumption. DNA quantification and purity estimation were performed using the NanoDropTM 1000 UV/VIS spectrophotometer and DNA integrity and PCR products were determined by gel-electrophoresis. DNA quantity ranged from 92.77 ± 11.52 to 5477.4 ± 22.03 ng/µL, with A260/280 from 1.14 ± 0.10 to 1.94 ± 0.16, and A260/230 0.37 ± 0.05 to 1.91 ± 0.17. There were also great differences in time consumption per sample, ranging from 1 hr 15 min to 7 hr 5 min. The determined costs per sample were ranging from 0.12 € to 2.29 € per sample. All tested methods were suitable for the isolation of A. flavus genomic DNA and subsequently for PCR reaction

Polymorphisms of Toll-like Receptors 2 and 4 in Chronically Infected Hepatitis C Patients from North-east Croatia

Chronic infection with hepatitis C virus (HCV) is caused by an inadequate immune response. Experimental data suggest that the impaired activation of Toll-like receptors (TLRs) 2 and 4 contributes to chronic infection. We assessed the distribution of three single-nucleotide polymorphisms (SNPs) in the TLR2 (Arg753Gln) and TLR4 (Asp299Gly/Thr399Ile) genes in individuals from north-east Croatia and their effect on the outcome of antiviral therapy. The study consisted of 60 chronically infected patients and 40 healthy subjects. TLR polymorphisms were determined by the PCR-based melting curve analysis. HCV genotyping was performed using the Linear Array Hepatitis C Virus Genotyping Test. Thirty-three patients were treated with standard interferon and ribavirin therapy, and their viral load was evaluated at weeks 28 and 53 after the beginning of therapy. The majority of chronic infections were caused by genotype 1 (77%), followed by genotypes 3 (15%) and 4 (7%). Patients with genotype 1 had higher viral loads than patients infected with other genotypes (P = 0.0428). Healthy individuals and patients with chronic infection had similar frequencies of TLR2-Arg753Gln and TLR4-Asp299Gly/Thr399Ile SNPs. Heterozygous and homozygous TLR4-Asp299Gly/Thr399Ile polymorphisms correlated with higher viral loads and delayed responses to antiviral therapy. We have provided the first evidence that TLR4 polymorphisms influence the success of antiviral therapy in our region. This suggests that therapeutic strategies should be adjusted not only according to HCV genotype but also to individual TLR polymorphism(s)