E. Arrese

Human Papillomavirus (HPV) genotype 18 variants in patients with clinical manifestations of HPV related infections in Bilbao, Spain

BackgroundHuman papillomavirus (HPV) variants differ in their biological and chemical properties, and therefore, may present differences in pathogenicity. Most authors classified variants based on the phylogenetic analysis of L1 region. Nevertheless, recombination in HPV samples is becoming a usual finding and thus, characterizing genetic variability in other regions should be essential.ObjectivesWe aimed to characterize the genetic variability of HPV 18 in 5 genomic regions: E6, E7, E4, L1 and the Upstream Regulatory Region (URR), working with both single infection and multiple HPV infection samples. Furthermore, we aimed to assess the prevalence of HPV 18 variants in our region and look for possible existence of recombination as well as analyze the relationship between these variants and the type of lesion.MethodsFrom 2007 to 2010, Clinical Microbiology and Infection Control Department analyzed 44 samples which were positive for HPV 18. Genetic variability was determined in PCR products and variants were assigned to European, Asian-amerindian or African lineage. Recombination and association of variants with different types of lesion was studied.ResultsGenetic analysis of the regions revealed a total of 56 nucleotide variations. European, African and Asian-amerindian variants were found in 25/44 (56.8%), 10/44 (22.7%) and 5/44 (11.4%) samples, respectively. We detected the presence of recombinant variants in 2/44 (4.5%) cases. Samples taken from high-grade squamous intraepithelial lesions (H-SIL) only presented variants with specific-african substitutions.ConclusionsMultiple HPV infection, non-european HPV variants prevalence and existence of recombination are considered risk factors for HPV persistence and progression of intraepithelial abnormalities, and therefore, should be taken into consideration in order to help to design and optimize diagnostics protocols as well as improve epidemiologic studies.Our study is one of the few studies in Spain which analyses the genetic variability of HPV18 and we showed the importance of characterizing more than one genomic region in order to detect recombination and classify HPV variants properly.

Comparison of INNO-LIPA and TRUGENE Assays for Genotyping and Drug-Resistance Mutations in Chronic Hepatitis B Virus Infection

Objective: Hepatitis B virus (HBV) genotyping and detection of resistance to drugs have become essential in epidemiological and clinical diagnosis. Our main objective was to determine the prevalence of HBV genotypes and drug-resistance mutations in chronic asymptomatic carriers and chronic HBV sufferers comparing 2 detection assays. Methods: Serum samples from 28 chronic HBV patients and 22 chronic asymptomatic carriers were analyzed. For HBV genotyping, the INNO-LIPA and TRUGENE™ HBV genotyping kits were evaluated. For drug-resistance mutations, INNO-LIPA DR v2 and INNO-LIPA DR v3 prototype and the TRUGENE™ HBV genotyping kit were evaluated. Results: In HBV genotyping, concordant results were 98% and both assays were able to detect more than one genotype. Different genotypes were detected, the most prevalent being D (46%) and A (26%). In relation to drug-resistance mutations, the sensitivity of the line probe assay was lower than TRUGENE because INNO-LIPA could not detect two mutations (S202G and V214A). Conclusions: Both assays are easy and suitable for detecting HBV genotype and drug-resistance mutations and for routine laboratory use. However, TRUGENE presented better sensitivity in both analyses and it is possible to conduct both on the same sample. This assay is also able to detect primary and secondary mutations.

P1.022 Human Papillomavirus 16 Variants Analysis in Multiple Infections

Background/Objectives Human papillomavirus type 16 (HPV 16) is the primary aetiology of cervical cancer. Risk factors associated to develop of malignant lesions include: infection persistence, specific HPV 16 variants and multiple infections presence. We had characterised the genomic variability of E6, E7 and L1 genes in HPV 16 multiple infection patients samples and analysed the relationship between intratypic variants and lesion grade. Methods HPV 16 multiple infection samples were amplified with three region type-specific primers and amplicons were sequenced using the “Big Dye Terminator Cycle Sequencing kit”. Sequences were aligned using Edit Sequence Alignment Editor and ClustalW, and compared with Genbank reported reference sequences: European (E), African (AF1 and AF2) and Asian-American (AA). Lesions were divided as negative, low-grade (L-SIL) or high-grade (H-SIL). Results HPV 16 multiple infections were identified in 125 samples and 78 of them were analysed for intratypic variations: 72 E variants (92.3%), 4 AA variants (5.1%), one AF1 (1.3%) and one AF2 variant (1.3%). In E6 region, missense mutations (A104del and T350G) were defined in 59% and 41% of samples. In E7 region, a mainly synonymous variation (G849A, 41.33%) was detected. In L1 region, non-synonymous replacements were only identified: 6901insCAT (30%), 6902 insATC (65.7%) and GAT6951del (97.1%). European variants were mainly detected in samples with no lesion while non-european variants were only found in H-SIL or L-SIL. Conclusions E6, E7 and L1 genes are useful to determinate among E, AA and AF1/AF2 variants. Non-european variants are also present in our population. Nucleotide variations different to define variants must be studied owing to their potential impact on pathogenesis. T350G nucleotide substitution is associated with elevated risk of cervical carcinomas. These variations should be taken into consideration. Funding: S-PC11BF002 project (Saiotek, Department of Industry, Basque Government).