Immune-escape mutations and stop-codons in HBsAg develop in a large proportion of patients with chronic HBV infection exposed to anti-HBV drugs in Europe.

BACKGROUND: HBsAg immune-escape mutations can favor HBV-transmission also in vaccinated individuals, promote immunosuppression-driven HBV-reactivation, and increase fitness of drug-resistant strains. Stop-codons can enhance HBV oncogenic-properties. Furthermore, as a consequence of the overlapping structure of HBV genome, some immune-escape mutations or stop-codons in HBsAg can derive from drug-resistance mutations in RT. This study is aimed at gaining insight in prevalence and characteristics of immune-associated escape mutations, and stop-codons in HBsAg in chronically HBV-infected patients experiencing nucleos(t)ide analogues (NA) in Europe. METHODS: This study analyzed 828 chronically HBV-infected European patients exposed to ≥ 1 NA, with detectable HBV-DNA and with an available HBsAg-sequence. The immune-associated escape mutations and the NA-induced immune-escape mutations sI195M, sI196S, and sE164D (resulting from drug-resistance mutation rtM204 V, rtM204I, and rtV173L) were retrieved from literature and examined. Mutations were defined as an aminoacid substitution with respect to a genotype A or D reference sequence. RESULTS: At least one immune-associated escape mutation was detected in 22.1% of patients with rising temporal-trend. By multivariable-analysis, genotype-D correlated with higher selection of ≥ 1 immune-associated escape mutation (OR[95%CI]:2.20[1.32-3.67], P = 0.002). In genotype-D, the presence of ≥ 1 immune-associated escape mutations was significantly higher in drug-exposed patients with drug-resistant strains than with wild-type virus (29.5% vs 20.3% P = 0.012). Result confirmed by analysing drug-naïve patients (29.5% vs 21.2%, P = 0.032). Strong correlation was observed between sP120T and rtM204I/V (P < 0.001), and their co-presence determined an increased HBV-DNA. At least one NA-induced immune-escape mutation occurred in 28.6% of patients, and their selection correlated with genotype-A (OR[95%CI]:2.03[1.32-3.10],P = 0.001). Finally, stop-codons are present in 8.4% of patients also at HBsAg-positions 172 and 182, described to enhance viral oncogenic-properties. CONCLUSIONS: Immune-escape mutations and stop-codons develop in a large fraction of NA-exposed patients from Europe. This may represent a potential threat for horizontal and vertical HBV transmission also to vaccinated persons, and fuel drug-resistance emergence.

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.

[Investigation of HIV-1 primary drug resistance mutations in antiretroviral therapy-naive cases]. / Antiretroviral tedavi almayan olgularda HIV-1 primer ilaç direnci mutasyonlarinin arastirilmasi

Following the report of the first AIDS case in Turkey in 1985, the total number of HIV-positive and AIDS cases is 6802 near the end of June 2013, according to the data obtained from the Turkish Ministry of Health. Although combined antiretroviral drug therapy has greatly improved the life-span and the life quality of the patients, HIV-1 drug resistance poses a major obstacle for treatment outcome. The aim of this study was to detect the presence of primary drug resistance in antiretroviral therapy-naive cases. Plasma samples obtained from 190 cases (143 male, 28 female, 19 unknown; age range: 20-72 yrs; mean age: 34.7 yrs) sent to AIDS Confirmation Center of Turkish Public Health Institution, from different provinces of Turkey (Ankara: 64; Adana: 42; Istanbul: 20; Antalya: 13; Gaziantep: 11; Erzurum: 5; Elazig: 3; Malatya: 3; Mersin: 3; and 26 samples from other 21 regions) for routine HIV-1 genotyping test between May 2010-April 2014 period were included in the study. In viral pol gene, complete protease encoding sequence and the first 335 codons of reverse transcriptase (RT) encoding sequence were analyzed by Sanger population-based sequencing method with a commercial test kit (ViroSeq, Abbott/Celera Diagnostics, USA). An alternative in house PCR method with different set of primers was used when the commercial test failed. Primary drug resistance mutations were identified according to the WHO 2009 drug resistance surveillance list. The mean HIV-RNA level at the time of resistance testing was 5.07 (range, 2.09-7.67) log10 copies/ml and the median CD4+ T cell count was 280.3 (range, 0-1000) cells/mm³. The period between the diagnosis of HIV infection and HIV-1 drug resistance test was 18.4 (range, 0-973) weeks. Most prevalent HIV-1 subtypes were subtype B (31%); recombinant B, F1 (24.7%), sub-subtype A1 (16.8%), recombinant B/CRF02_AG (10.5%) and CRF02_G (7.8%). Analysis of our results showed that 10% (19/190) of the samples exhibited resistance mutations. Detected mutations were as follows: M41L, K70E, M184V, L210W and T215C/D/S, responsible for nucleoside RT inhibitor (NRTI) resistance; K103N/S and Y181C, responsible for non-nucleoside RT inhibitor (NNRTI) resistance; M46L and L90M, responsible for protease inhibitor (PI) resistance. NRTI, NNRTI and PI mutation rates in the samples were found as 5.2%, 3.1% and 2.1%, respectively. Both NRTI and NNRTI mutations were detected in one sample. Two mutations leading to NRTI resistance were obtained together in five samples. Seven out of the 19 strains with primary resistance mutation were isolated from samples sent from Ankara, three from Adana, two of each from Istanbul, Erzurum and Mersin, and one of each from Amasya, Samsun and Tokat provinces. There were no statistically significant differences in terms of age, gender, CD4⁺ T cell count, time from diagnosis to resistance testing between the patient group with primary drug resistance and the group without resistance (p> 0.05). However, the mean HIV-RNA level in patients with primary drug resistance [4.77 (2.95-6.87) log(10) copies/ml] was significantly lower than those without primary drug resistance [5.11 (2.09-7.67) log10 copies/ml] (p= 0.043). Our results revealed a relatively high (10%) prevalence of HIV-1 primary drug resistance. We therefore support the need for routine HIV resistance testing so that clinicians can individualize their treatments taking into account the presenting drug resistance.

Evolutionary dynamics of HBV-D1 genotype epidemic in Turkey.

Hepatitis B virus (HBV), is the leading cause of liver diseases infecting an estimated 240 million persons worldwide. The HBV prevalence rates are variables between different countries, with an high level of endemicity in the south-eastern part of Europe. Seven main HBV-D subgenotypes have been described until now (D1-D7). Turkey, seems to have played an important role in the penetration of HBV-D1 in the Mediterranean area. The importance of Turkey in the European epidemiology of HBV is also suggested by the observation that the highest spread of HBV infection in the Continent are reported in Turkey with Romania, Bulgaria, Greece, Albania and some southern regions of Italy. In this paper the molecular epidemiology and the epidemiological history of HBV-D in Turkey was studied, by characterizing 34 new Turkish isolates and performing a phylogeographic reconstruction. By using a phylodynamic and phylogeographic Bayesian approach, the analysis suggested that HBV-D1 originated in Turkey about in the early 1940s. The large prevalence of D1 in comparison to the other subgenotypes in Turkey confirms the importance of this Country as epidemiological reservoir of HBV-D1 dispersion. The phylogeny suggests that after each initial introduction of the virus in a specific population, separate transmission clusters have been evolving along independent phylogenetic lineages. Better characterization and continuous monitoring of such groups are going to be crucial to understand in detail the epidemiology of HBV-D1 subgenotype in Turkey and to assess the efficacy of prevention, vaccination and therapy in controlling the epidemic.

Reconstruction of the evolutionary dynamics of the hepatitis C virus 1b epidemic in Turkey.

Worldwide, 12.5% of the more than 170 million people infected by hepatitis C virus (HCV), live in Eastern Mediterranean countries. In Turkey, the prevalence of HCV infection ranges from 0.3% to 0.4% of the general population. We investigated the distribution of HCV subtype 1b in Turkey by analysing the NS5b viral genomic region, using a Bayesian coalescent-based framework and phylogeographical analysis to estimate the origin of the HCV 1b subtype epidemic and the genetic diversification of the virus in Turkey. The dataset consisted of 24 NS5b sequences obtained from patients chronically infected with HCV subtype 1b admitted to the different health districts of Ankara hospital plus the reference sequences for phylogenetic analysis. An independent dataset including the same 342-nt NS5b fragment from all over the world (203 sequences) was used to calibrate the evolutionary rate. Using the relaxed clock model, we estimated a mean evolutionary rate of 0.84 × 10(-3) sub/site/year (95% highest posterior density interval HPD 0.16-1.5 × 10(-3)). The results of the phylogeographical analysis suggested that the HCV epidemic probably originated in Greece during the first decade of 1900 and, a few years later (in the 1920s or 1930s), successfully spread to neighboring countries such as Turkey and Cyprus. The clustering of the majority of the Turkish strains in a single monophyletic group suggests the subsequent segregated circulation of the virus in the country during the years 1940-1999, which was probably due to unsafe medical parenteral procedures, with drug addiction playing a relatively negligible role. The Bayesian skyline plot (BSP) showed a growth in the number of effective infections between the 1940s and the 1990s, when the curve reached a plateau that still remains today, suggesting a partial success of improved transfusional policies. A coalescent-based approach to population dynamics can improve our understanding of the origin and spread of epidemics in a limited geographical area.

[Investigation of Leishmania infantum seropositivity in HIV/AIDS patients]. / HIV/AIDS hastalarinda Leishmania infantum seropozitifliginin arastirilmasi.

In recent years an increase in the rate of detection of HIV and Leishmania co-infections has been reported from many countries especially countries in Southern Europe. Visceral leishmaniasis (VL) is sporadically detected in some parts of Turkey. Although the natural transmission is via sandfly bites, VL may be transmitted by needle sharing of intravenous drug addicts or by blood transfusion in HIV/AIDS patients. The aim of this study was to investigate the presence of specific antibodies against Leishmania infantum, which is the causative agent of VL, in the sera of HIV/AIDS patients. A total of 79 HIV/AIDS patients (61 male, 18 female; mean age: 30 +/- 2 years) with confirmed diagnosis by HIV Reference Laboratory of Refik Saydam Hygiene Center between the years of 2004-2006, were included in the study. L. infantum antibodies were searched by fast agglutination screening test (FAST), direct agglutination test (DAT), indirect immunofluorescent antibody test (IFAT) and rK39 dipstick assay. Only one serum sample (1.2%) was found to be seropositive by all of the serological tests (> 1/100 by FAST, 1/3200 by DAT, 1/256 by IFAT, and specific bands for L. infantum by rK39 dipstick test), while the remaining samples were negative with all of the methods. The seropositive serum was from a 49 years-old heterosexual male, living on the Mediterranean cost and has had acquired the HIV infection by sexual contact. He has no history of intravenous drug use but he had experienced blood transfusion. Since the seropositive serum sample was collected 2-3 weeks after the transfusion, the transmission of L. infantum was thought to be during blood transfusion, however it could also be acquired via a previous sandfly bite. In conclusion although the rate of L. infantum seropositivity was low in HIV/AIDS patients in our study, the possibility of HIV/Leishmania co-infections should be considered.

[Sensitivities of various cell cultures for the isolation of enteroviruses]. / Enteroviruslarin izolasyonunda degisik hücre kültürlerinin duyarliliklari.

In the present study, the sensitivities of HEp-2 (human epithelioma), RD (rhabdomyosarcoma) and L20B (mouse cells, that have receptors for human polioviruses) cell cultures have been evaluated and compared, for the isolation and identification of enteroviruses from the stool and cerebrospinal fluid samples of patients with acute flask paralysis and aseptic meningitis, which were examined between the years 1999-2000, in Refik Saydam Institute of Hygiene Center, Virology, Tissue Culture and Enterovirus Laboratory. Of a total of 1663 samples, 131 viral strains were isolated, and 120 of them were identified as enteroviruses, and 11 as adenoviruses. The isolation rates of 48 Sabin-like polioviruses from HEp-2, RD and L20B cell lines were found similar, as 83.3%, 87.5% and 91.6%, respectively. All of 47 Echovirus strains were isolated from RD cells, all of 13 Coxsackie type B strains were isolated from HEp-2 cells, and all of 12 non-polio enteroviruses were isolated from RD cells. All of 11 adenovirus strains that have been grown in Hep-2 cells, were thought to be occasionally isolated due to the passage of viruses to gastrointestinal tract, and excreted via stool, thus having no clinical significance for these patients. As a result, it was concluded that, all of these three cell lines and especially L20B were sensitive for polioviruses, RD cell line being more sensitive for Echovirus, and HEp-2 cell line being more sensitive for Coxsackie type B virus strains.

[Standardization of neutralization tests using the COBL cell line and comparison with the particle agglutination test for measles serology]. / Kizamik serolojisinde nötralizasyon testinin COBL Hücresi kullanilarak standardizasyonu ve partikül aglütinasyon testi ile karsilastirilmasi.

The aim of the present study was the detection and comparison of measles antibody titers with particle agglutination (PA) and neutralization (Nt) methods, in the sera samples of 364 subjects from different age groups. PA method was performed with a commercial test kit (Serodiameasles, Fujirebio Com. Japan), and Nt test which was standardized in this study, by using COBL (cord blood) cell lines, has been started to use in our laboratory as a reference method. As a result, antibody titers detected by PA were in parallel to the titers which detected by Nt test, and it was concluded that the differences in antibody titers would arise from the differences of test principles and viral antigens.