Maja M. Lunar

Hepatitis C virus genotypes in 1,504 patients in Slovenia, 1993-2007.

In order to identify the main routes of hepatitis C (HCV) transmission and to determine the HCV genotype distribution and its dynamics during a 15‐year period in Slovenia, HCV genotypes were detected using the INNO‐LiPA HCV II (Innogenetics) test for serum samples obtained from 1,504 patients representing 72.6% of all patients with chronic hepatitis C diagnosed from 1993 to 2007. HCV genotype 1 was predominant (56%), followed by genotypes 3, 2, and 4, with a prevalence of 37.8%, 5%, and 1.2%, respectively. HCV genotypes 5 and 6 were not detected in any patient. Patients infected with HCV genotype 3 were significantly younger (mean age 28.9 ± 8.5 years) than those infected with genotype 1 (mean age 38.9 ± 14.8 years; P < 0.0001) and those infected with HCV genotype 2 (mean age 50.3 ± 18.2 years; P < 0.0001). Intravenous drug use was identified as the most frequent possible HCV transmission route (34.3%), followed by medical‐related transmission such as transfusion of HCV‐contaminated blood or blood products, and hemodialysis (12.5%). Being an intravenous drug user was found to be strongly associated with HCV genotype 3 (OR, 3.71 [95% CI, 2.97–4.65]; P < 0.0001) and reporting infection by transfusion of blood or blood products was found to be strongly associated with HCV genotype 1 (OR, 3.28 [95% CI, 2.18–4.95]; P < 0.0001). During the 15‐year period, the proportion of genotype 3 increased substantially, reflecting the fact that the HCV epidemic in Slovenia is driven mostly by intravenous drug use. J. Med. Virol. 81:634–639, 2009

Comparative Performances of HIV-1 RNA Load Assays at Low Viral Load Levels: Results of an International Collaboration

ABSTRACT Low-level viremia during antiretroviral therapy and its accurate measurement are increasingly relevant. Here, we present an international collaboration of 4,221 paired blood plasma viral load (pVL) results from four commercial assays, emphasizing the data with low pVL. The assays compared were the Abbott RealTime assay, the Roche Amplicor assay, and the Roche TaqMan version 1 and version 2 assays. The correlation between the assays was 0.90 to 0.97. However, at a low pVL, the correlation fell to 0.45 to 0.85. The observed interassay concordance was higher when detectability was defined as 200 copies/ml than when it was defined as 50 copies/ml. A pVL of ∼100 to 125 copies/ml by the TaqMan version 1 and version 2 assays corresponded best to a 50-copies/ml threshold with the Amplicor assay. Correlation and concordance between the viral load assays were lower at a low pVL. Clear guidelines are needed on the clinical significance of low-level viremia.

Distribution of human papillomavirus genotypes in women with cervical cancer in Slovenia

OBJECTIVE The purpose of the present study was to establish the distribution of human papillomavirus (HPV) genotypes in a representative population of women with cervical cancer in Slovenia in order to contribute to the lacking data on HPV in cervical cancer and to assess the potential local benefit of future prophylactic HPV vaccination. STUDY DESIGN A total of 284 samples of cervical cancer were analyzed including archival samples, cervical scrapes and fresh tissue samples. Polymerase chain reaction with GP5+/GP6+ primers was performed in all samples for HPV deoxyribonucleic acid (DNA) detection. All GP5+/GP6+ negative samples were additionally tested using CPI/CPIIg primers and INNO-LiPA HPV genotyping assay. RESULTS After exclusion of 6 samples with unsuccessful amplification of beta-globin gene, 262 of 278 cervical cancer samples (94.2%) were HPV DNA positive. HPV genotypes found in the decreasing order of frequency were: HPV 16 (64.9%), HPV 18 (12.2%), HPV 33 (4.7%), HPV 45 (4.1%), followed by HPV 31, 51, 58, 59, 35, 52, 73 and 82 (3.5-0.2%). HPV positive samples were more frequent among squamous cell carcinomas than among adenocarcinomas/adenosquamous carcinomas (95.8% versus 85.5%; P=0.003). HPV 16 was more frequently found in squamous cell carcinomas than in adenocarcinomas/adenosquamous carcinomas (69.9% versus 37.5%; P<0.001), while the opposite was true for HPV 18 (6% versus 41.7%; P<0.001). CONCLUSION Prophylactic HPV vaccination with currently available vaccines could prevent up to 77.1% of cervical cancer in Slovenia, which is caused by HPV 16 or HPV 18.

Human Papillomavirus Genotype Specificity of Hybrid Capture 2 Low-Risk Probe Cocktail

ABSTRACT A genotyping study of 285 Hybrid Capture 2 low-risk probe cocktail-positive specimens showed cross-reactivity with several untargeted human papillomavirus genotypes. Cross-reactivity was often clinically beneficial due to the detection of untargeted low-risk genotypes. A total of 8.4% of positive results, usually weak, were due to cross-reactivity with high-risk genotypes. Establishment of a gray zone is recommended.

Digene HPV Genotyping RH Test RUO: Comparative evaluation with INNO-LiPA HPV Genotyping Extra Test for detection of 18 high-risk and probable high-risk human papillomavirus genotypes

BACKGROUND Standardized and validated methods for the specific detection and identification of a spectrum of high-risk (hr) HPV genotypes will be necessary if HPV genotyping gains an important role in the clinical management of HPV-related precancerous lesions and cancers. OBJECTIVES The first comparative evaluation of novel HPV genotyping Digene HPV Genotyping RH Test RUO (Qiagen, Hilden, Germany) with standard INNO-LiPA HPV Genotyping Extra CE assay (Innogenetics, Gent, Belgium). STUDY DESIGN Seventy hr-HPV positive samples were tested in parallel with both genotyping assays. The results were interpreted taking into account 15 hr-HPV and 3 probable hr-HPV genotypes that can be identified by both assays (assay-common genotypes). RESULTS Concordant results (a complete match of assay-common genotypes or negative using both assays) and compatible results (at least one genotype in common) were obtained in 42 (60.0%) and 28 (40.0%) samples, respectively. No discordant results for assay-common genotypes were obtained. Of 42 samples with compatible results, the presence of at least one assay-common genotype was detected in 37 samples, while no HPV was detected in two samples by both assays and only a single low-risk HPV was detected by INNO-LiPA in three samples. CONCLUSIONS A novel Digene test is suitable for the detection of hr-HPV genotypes in clinical samples and it provides comparable results to the well established INNO-LiPA assay. Although INNO-LiPA identified significantly more samples with multiple HPV genotypes than the Digene test, the clinical benefit of such a difference is at present unclear.

Combined Analysis of the Prevalence of drug Resistant Hepatitis B Virus in antiviral therapy Experienced patients in Europe (CAPRE)

BACKGROUND European guidelines recommend treatment of chronic hepatitis B virus infection (CHB) with the nucleos(t)ide analogs (NAs) entecavir or tenofovir. However, many European CHB patients have been exposed to other NAs, which are associated with therapy failure and resistance. The CAPRE study was performed to gain insight in prevalence and characteristics of NA resistance in Europe. METHODS A survey was performed on genotypic resistance testing results acquired during routine monitoring of CHB patients with detectable serum hepatitis B virus DNA in European tertiary referral centers. RESULTS Data from 1568 patients were included. The majority (73.8%) were exposed to lamivudine monotherapy. Drug-resistant strains were detected in 52.7%. The most frequently encountered primary mutation was M204V/I (48.7%), followed by A181T/V (3.8%) and N236T (2.6%). In patients exposed to entecavir (n = 102), full resistance was present in 35.3%. Independent risk factors for resistance were age, viral load, and lamivudine exposure (P < .001). CONCLUSIONS These findings support resistance testing in cases of apparent NA therapy failure. This survey highlights the impact of exposure to lamivudine and adefovir on development of drug resistance and cross-resistance. Continued use of these NAs needs to be reconsidered at a pan-European level.

Persistence of frequently transmitted drug-resistant HIV-1 variants can be explained by high viral replication capacity

BackgroundIn approximately 10% of newly diagnosed individuals in Europe, HIV-1 variants harboring transmitted drug resistance mutations (TDRM) are detected. For some TDRM it has been shown that they revert to wild type while other mutations persist in the absence of therapy. To understand the mechanisms explaining persistence we investigated the in vivo evolution of frequently transmitted HIV-1 variants and their impact on in vitro replicative capacity.ResultsWe selected 31 individuals infected with HIV-1 harboring frequently observed TDRM such as M41L or K103N in reverse transcriptase (RT) or M46L in protease. In all these samples, polymorphisms at non-TDRM positions were present at baseline (median protease: 5, RT: 6). Extensive analysis of viral evolution of protease and RT demonstrated that the majority of TDRM (51/55) persisted for at least a year and even up to eight years in the plasma. During follow-up only limited selection of additional polymorphisms was observed (median: 1).To investigate the impact of frequently observed TDRM on the replication capacity, mutant viruses were constructed with the most frequently encountered TDRM as site-directed mutants in the genetic background of the lab strain HXB2. In addition, viruses containing patient-derived protease or RT harboring similar TDRM were made. The replicative capacity of all viral variants was determined by infecting peripheral blood mononuclear cells and subsequently monitoring virus replication. The majority of site-directed mutations (M46I/M46L in protease and M41L, M41L + T215Y and K103N in RT) decreased viral replicative capacity; only protease mutation L90M did not hamper viral replication. Interestingly, most patient-derived viruses had a higher in vitro replicative capacity than the corresponding site-directed mutant viruses.ConclusionsWe demonstrate limited in vivo evolution of protease and RT harbouring frequently observed TDRM in the plasma. This is in line with the high in vitro replication capacity of patient-derived viruses harbouring TDRM compared to site-directed mutant viruses harbouring TDRM. As site-directed mutant viruses have a lower replication capacity than the patient-derived viruses with similar mutational patterns, we propose that (baseline) polymorphisms function as compensatory mutations improving viral replication capacity.

Low prevalence of hepatitis C infection among HIV-infected individuals in Slovenia: a nationwide study, 1985–2013

After introduction of highly active antiretroviral therapy and consecutive successful control of HIV infection hepatitis C virus (HCV) has become an important pathogen in HIV infected patients. HIV infection in a person who is also HCV infected results in reduced rate of spontaneous HCV RNA clearance, faster liver disease progression and more aggressive course of liver disease. A total of 639 individuals were cumulatively reported as HIV-infected in Slovenia until the end of 2013. The majority of HIV-infected were men (553/639; 86.5%) and among them 68.2% were men who have sex with men. The predominant HIV-1 subtype in Slovenia is subtype B, which is present in 85.8% of the infected individuals. We tested 575 (90.0%) of 639 Slovenian individuals who were confirmed as HIV positive by the end of 2013 for HCV infection. All individuals included in a study were tested for both anti-HCV and HCV RNA. Out of 575 HIV-infected individuals 44 (7.6%) had anti-HCV specific antibodies, and 32 of them (72.7%) were also HCV RNA positive. We didn’t detect HCV RNA alone in any of the 531 anti-HCV-negative individuals. Anti-HCV positivity was significantly more frequent in HIV-infected individuals who acquired HIV by parenteral route (73.3%) comparing with those who acquired HIV by sexual route (2.6%). The most prevalent HCV genotype among HIV-infected individuals was genotype 1 (70.8%), followed by genotype 3 (16.7%), genotype 4 (8.3%) and genotype 2 (4.2%). HCV genotypes distribution didn’t significantly differ between HIV-positive and HIV-negative, HCV-positive Slovenian patients. Our study which was performed on the highest proportion per entire population of HIV-infected individuals from a certain country identified Slovenia as the country with the lowest prevalence of HCV infection among HIV-infected individuals. The predominance of sexual transmission of HIV (79.2%) in Slovenia and the fact that HIV has not yet entered the intravenous drug users’ community in Slovenia are the two most likely reasons for low prevalence of HIV-HCV co-infection. However, the present epidemiological situation in Slovenia needs to be monitored closely since it could quickly change in a case of an increase in the incidence of acute hepatitis C among HIV-infected men who have sex with men and/or increase of HIV infection among intravenous drug users in the country, as it happened recently in some neighboring countries.

Prevention of human papillomavirus (HPV)-related tumors in people living with human immunodeficiency virus (HIV)

ABSTRACT Introduction: In comparison to their HIV-negative counterparts, people living with HIV (PLWH) have a higher prevalence of human papillomavirus (HPV) infection in various anatomical sites coupled with increased HPV persistence, higher risk of HPV-related tumors, and faster disease progression. Areas covered: Gender-neutral prevention strategies for HPV-related cancers in PLWH discussed: ABC approach, HPV vaccination, antiretroviral treatment (ART), anal cancer screening, and smoking cessation. Gender specific strategies: cervical cancer screening reduces the incidence and mortality of cervical cancer and circumcision might reduce the risk of HPV infections in men. Expert commentary: HPV-related cancer incidence has not declined (e.g. cervical cancer) and has even increased (e.g. anal cancer) in the ART era, demanding an effective HPV prevention strategy. HPV vaccination should be introduced into national prevention programs worldwide immediately because current prophylactic vaccines are safe, tolerable, and immunogenic in PLWH. HPV vaccine efficacy trials in PLWH are essential to determine the most appropriate immunization schedule. The population most at risk of anal cancer is HIV-positive men who have sex with men, who are not protected by herd immunity if only the female population is vaccinated. Unvaccinated PLWH need enhanced surveillance for early detection of HPV-related cancers and their precursors.

Longitudinal trends of recent HIV-1 infections in Slovenia (1986–2012) determined using an incidence algorithm

Resolving dilemma whether the rise in the number of HIV diagnoses represents an actual increase in HIV transmissions or is a result of improved HIV surveillance is crucial before implementing national HIV prevention strategies. Annual proportions of recent infections (RI) among newly diagnosed persons infected with HIV‐1 in Slovenia during 27 years (1986–2012) were determined using an algorithm consisting of routine baseline CD4 and HIV viral load measurements and the Aware BED EIA HIV‐1 Incidence Test (BED test). The study included the highest coverage of persons diagnosed with HIV during the entire duration of an HIV epidemic in a given country/region (71%). Out of 416 patients, 170 (40.9%) had a baseline CD4 cell count less than 200 cells/mm3 and/or HIV‐1 viral load less than 400 copies/ml and were characterized as having a long‐standing infection (LSI). The remaining 246 patients were additionally tested using the BED test. Overall, 23% (97/416) of the patients were labeled RI. The characteristics significantly associated with RI were as follows: younger age, acute retroviral syndrome, CDC class A and other than C, no AIDS defining illnesses, HIV test performed in the past, a higher viral load, and a higher CD4 cell count. An interesting trend in the proportion of RI was observed, with a peak in 2005 (47% of RI) and the lowest point in 2008 (12%) in parallel with a rise in the numbers of new HIV diagnoses. This study could help promote the idea of introducing periodic HIV incidence monitoring using a simple and affordable algorithm. J. Med. Virol. 87:1510–1516, 2015.