Neuroprotective Properties of Oleanolic Acid-Computational-Driven Molecular Research Combined with In Vitro and In Vivo Experiments.

Oleanolic acid (OA), as a ubiquitous compound in the plant kingdom, is studied for both its neuroprotective and neurotoxic properties. The mechanism of acetylcholinesterase (AChE) inhibitory potential of OA is investigated using molecular dynamic simulations (MD) and docking as well as biomimetic tests. Moreover, the in vitro SH-SY5Y human neuroblastoma cells and the in vivo zebrafish model were used. The inhibitory potential towards the AChE enzyme is examined using the TLC-bioautography assay (the IC50 value is 9.22 µM). The CH-π interactions between the central fragment of the ligand molecule and the aromatic cluster created by the His440, Phe288, Phe290, Phe330, Phe331, Tyr121, Tyr334, Trp84, and Trp279 side chains are observed. The results of the in vitro tests using the SH-SY5Y cells indicate that the viability rate is reduced to 71.5%, 61%, and 43% at the concentrations of 100 µg/mL, 300 µg/mL, and 1000 µg/mL, respectively, after 48 h of incubation, whereas cytotoxicity against the tested cell line with the IC50 value is 714.32 ± 32.40 µg/mL. The in vivo tests on the zebrafish prove that there is no difference between the control and experimental groups regarding the mortality rate and morphology (p > 0.05).

Polymorphisms Related to Iron Homeostasis Associate with Liver Disease in Chronic Hepatitis C.

Dysregulation of iron metabolism in chronic hepatitis C (CHC) is a significant risk factor for hepatic cirrhosis and cancer. We studied if known genetic variants related to iron homeostasis associate with liver disease progression in CHC. Retrospective analysis included 249 CHC patients qualified for antiviral therapy between 2004 and 2014. For all patients, nine SNPs within HFE, TFR2, HDAC2, HDAC3, HDAC5, TMPRSS6, and CYBRD1 genes were genotyped. Expression of selected iron-related genes, was determined with qRT-PCR in 124 liver biopsies, and mRNA expression of co-inhibitory receptors (PD-1, Tim3, CTLA4) was measured in 79 liver samples. CYBRD1 rs884409, HDAC5 rs368328, TFR2 rs7385804, and TMPRSS6 rs855791 associated with histopathological changes in liver tissue at baseline. The combination of minor allele in HDAC3 rs976552 and CYBRD1 rs884409 linked with higher prevalence of hepatocellular carcinoma (HCC) during follow up (OR 8.1 CI 2.2-29.2; p = 0.001). Minor allele in HDAC3 rs976552 associated with lower hepatic expression of CTLA4. Tested polymorphisms related to iron homeostasis associate with histopathological changes in the liver. The presence of both HDAC3 rs976552 G and CYBRD1 rs884409 G alleles correlates with HCC occurrence, especially in the group of patients with elevated AST (>129 IU/L). rs976552 in HDAC3 could impact immunological processes associated with carcinogenesis in CHC.

Proteomic response of A549 lung cancer cell line to protein-polysaccharide complex Venetin-1 isolated from earthworm coelomic fluid.

Earthworms' celomic fluid has long attracted scientists' interest due to their toxic properties. It has been shown that the elimination of coelomic fluid cytotoxicity to normal human cells was crucial for the generation of the non-toxic Venetin-1 protein-polysaccharide complex, which exhibits selective activity against Candida albicans cells as well as A549 non-small cell lung cancer cells. To find the molecular mechanisms behind the anti-cancer properties of the preparation, this research investigated the proteome response of A549 cells to the presence of Venetin-1. The sequential window acquisition of all theoretical mass spectra (SWATH-MS) methodology was used for the analysis, which allows for a relative quantitative analysis to be carried out without radiolabelling. The results showed that the formulation did not induce significant proteome responses in normal BEAS-2B cells. In the case of the tumour line, 31 proteins were up regulated, and 18 proteins down regulated. Proteins with increased expression in neoplastic cells are mainly associated with the mitochondrion, membrane transport and the endoplasmic reticulum. In the case of altered proteins, Venetin-1 interferes with proteins that stabilise the structures, i.e., keratin, glycolysis/gluconeogenesis and metabolic processes.

Polymorphisms within DIO2 and GADD45A genes increase the risk of liver disease progression in chronic hepatitis b carriers.

The study enrolled 284 patients with chronic hepatitis B virus infection. Participants included people with mild fibrotic lesions (32.5%), moderate to severe fibrotic lesions (27.5%), cirrhotic lesions (22%), hepatocellular carcinoma (HCC) in 5%, and people with no fibrotic lesions in 13%. Eleven SNPs within DIO2, PPARG, ATF3, AKT, GADD45A, and TBX21 were genotyped by mass spectrometry. The rs225014 TT (DIO2) and rs10865710 CC (PPARG) genotypes were independently associated with susceptibility to advanced liver fibrosis. However, cirrhosis was more prevalent in individuals with the GADD45A rs532446 TT and ATF3 rs11119982 TT genotypes. In addition, the rs225014 CC variant of DIO2 was more frequently found in patients with a diagnosis of HCC. These findings suggest that the above SNPs may play a role in HBV-induced liver damage in a Caucasian population.

Novel Venetin-1 nanoparticle from earthworm coelomic fluid as a promising agent for the treatment of non-small cell lung cancer.

The present research shows the antitumor activity of a protein-polysaccharide complex Venetin-1 obtained from the coelomic fluid of Dendrobaena veneta earthworms against A549 cancer cells. The investigations are a continuation of experiments on the antitumor activity of coelomic fluid obtained from this species. The Venetin-1 nanoparticle was obtained after thermal treatment of the coelomic fluid, separation from coelomocytes, filtration, and lyophilization. The preparation showed a selective effect on cancer cells, whereas normal cells were unaffected. Venetin-1 was effective against the lung cancer cells at doses of 31.3 and 62.5 µg/ml, and the results were imaged using light microscopy and scanning electron microscopy (SEM). The cells died mainly via the apoptosis pathway. Necrotic cells appeared sporadically in the microscopic view. SEM imaging revealed complete destruction of the A549 cells after the incubation with Venetin-1. The atomic force microscopy (AFM) analyses showed changes in the topography, peak force error images, and Young's modulus (elasticity) of the A549 cells after the incubation with Venetin-1. The transmission electron cryomicroscopy (Cryo-TEM) analysis indicated a polymeric nature of the analyzed preparation. The samples of Venetin-1 showed a very homogeneous size profile with the microparticle size of approximately 58.23 nm. A significant decrease in Venetin-1 binding to sphingomyelin was observed. Venetin-1 lost its pore-forming activity or deactivation of the pore-forming activity occurred. This confirms the absence of hemolytic capacity of Venetin-1 towards red blood cells. The conducted analyses show the suitability of the obtained complex for biomedical research. The next step will consist in analyses of the effect of Venetin-1 on the immune system in mice.

(Re-)activity in the caregiving situation: Genetic diversity within Oxytocin-Vasopressin Pathway is associated with salivary oxytocin and vasopressin concentrations in response to contact with a crying infant-simulator.

Oxytocin (OT) and vasopressin (AVP) hormones as well as their receptors (OXTR and AVPR1a) have been deemed crucial for caregiving and sensitive responsiveness to infant cues. However, previous research on genetic polymorphisms and OT and AVP levels in the context of caregiving were sparse and have brought contradictory findings. The aim of this reported observational study was to examine the impact of genetic variations within genes related to OT and AVP signaling pathway on hormones levels' changes in response to the caregiving situation. A total of 221 adult intimate couples (110 childless, non-pregnant and 111 expectant couples) participated in three 10 min sessions, during which they were taking care of a crying life-like simulator. 30 min prior to the first session salivary samples to analyze basal OT and AVP, and polymorphisms in OXTR, AVPR1a and CD38 genes were collected. Subsequent OT and AVP levels were measured 15 min after each session. The two most frequently studied OXTR SNPs (rs53576 and rs2254298) had no or a minor impact on higher OT levels, which were linked to rs1042778, rs13316193, rs2228485, rs2268490, rs4686302 genotypes. AVP levels were affected by rs1042778, rs13316193, rs4686302 and rs237887. OT levels varied depending on the OT (rs2770378, rs4813625), CD38 (rs379686), and 5-HTR2A (rs6314) genotype. OT and AVP levels were also associated with rs6314 (5-HTR2A). AVP levels were linked to ESR1 (rs1884051) and SIM1 (rs3734354) variations. Shorter variants of RS3 and RS1 were associated with lower levels of AVP. In conclusion, analyzed polymorphisms were associated with both the level and changes in OT and AVP hormone levels in the standardized situation of caregiving reactions to infant crying.

Empathy and Hormonal Changes as Predictors of Sensitive Responsiveness towards Infant Crying: A Study Protocol.

Sensitive responsiveness refers to parents' ability to recognize and respond to infants' cues and has been linked to parental empathy. Additionally, oxytocin (OT) and vasopressin (AVP) are hormones important for sensitivity and empathy. The aim of this study is to test the links between dispositional empathy along with changing OT and AVP levels and responsiveness to a life-like doll in couples and to verify whether these factors are predictors of responsiveness to a child's cues. Exploratory analyses include predictors of sensitive responsiveness: polymorphisms of OXTR, AVPR1a and CD38 genes, personal characteristics and relational factors. The project employs standardized experimental settings that can be used with non-parents and the assessment of parental sensitive responsiveness towards their child. The participants are couples expecting their first child (111) and childless couples (110). The procedure involves caretaking of a life-like doll. Salivary samples and questionnaire data are collected in a planned manner. In the second part, the expectant couples are invited for the assessment of their sensitivity to their own child (Free Play episodes). Parental sensitivity is assessed using the Ainsworth Sensitivity Scale. This paper presents an interdisciplinary research project that reaches beyond the questionnaire measurement, considering many factors influencing the dynamics of adult-infant interaction.

Superiority of MALDI-TOF Mass Spectrometry over Real-Time PCR for SARS-CoV-2 RNA Detection.

At present, the RT-PCR test remains the gold standard for early diagnosis of SARS-CoV-2. Nevertheless, there is growing evidence demonstrating that this technique may generate false-negative results. Here, we aimed to compare the new mass spectrometry-based assay MassARRAY® SARS-CoV-2 Panel with the RT-PCR diagnostic test approved for clinical use. The study group consisted of 168 suspected patients with symptoms of a respiratory infection. After simultaneous analysis by RT-PCR and mass spectrometry methods, we obtained discordant results for 17 samples (10.12%). Within fifteen samples officially reported as presumptive positive, 13 were positive according to the MS-based assay. Moreover, four samples reported by the officially approved RT-PCR as negative were positive in at least one MS assay. We have successfully demonstrated superior sensitivity of the MS-based assay in SARS-CoV-2 detection, showing that MALDI-TOF MS seems to be ideal for the detection as well as discrimination of mutations within the viral genome.

Liver Cirrhosis in Chronic Hepatitis B Patients Is Associated with Genetic Variations in DNA Repair Pathway Genes.

Liver cirrhosis (LC), contributing to more than 1 million of deaths annually, is a major healthcare concern worldwide. Hepatitis B virus (HBV) is a major LC etiological factor, and 15% of patients with chronic HBV infection (CHB) develop LC within 5 years. Recently, novel host genetic determinants were shown to influence HBV lifecycle and CHB course. DNA repair enzymes can affect dynamics of liver damage and are involved in HBV covalently closed circular DNA (cccDNA) formation, an essential step for viral replication. This study aimed to evaluate the possible role of genes representing key DNA-repair pathways in HBV-induced liver damage. MALDI-TOF MS genotyping platform was applied to evaluate variations within XRCC1, XRCC4, ERCC2, ERCC5, RAD52, Mre11, and NBN genes. Apart from older age (p < 0.001), female sex (p = 0.021), portal hypertension (p < 0.001), thrombocytopenia (p < 0.001), high HBV DNA (p = 0.001), and high aspartate aminotransferase (AST) (p < 0.001), we found that G allele at rs238406 (ERCC2, p = 0.025), T allele at rs25487 (XRCC1, p = 0.012), rs13181 GG genotype (ERCC2, p = 0.034), and C allele at rs2735383 (NBN, p = 0.042) were also LC risk factors. The multivariate logistic regression model showed that rs25487 CC (p = 0.005) and rs238406 TT (p = 0.027) were independently associated with lower risk of LC. This study provides evidence for the impact of functional and potentially functional variations in key DNA-repair genes XRCC1 and ERCC2 in HBV-induced liver damage in a Caucasian population.

Recent Advances in Understanding, Diagnosing, and Treating Hepatitis B Virus Infection.

Chronic hepatitis B virus (HBV) infection affects 292 million people worldwide and is associated with a broad range of clinical manifestations including cirrhosis, liver failure, and hepatocellular carcinoma (HCC). Despite the availability of an effective vaccine HBV still causes nearly 900,000 deaths every year. Current treatment options keep HBV under control, but they do not offer a cure as they cannot completely clear HBV from infected hepatocytes. The recent development of reliable cell culture systems allowed for a better understanding of the host and viral mechanisms affecting HBV replication and persistence. Recent advances into the understanding of HBV biology, new potential diagnostic markers of hepatitis B infection, as well as novel antivirals targeting different steps in the HBV replication cycle are summarized in this review article.

Identification of Selected Antibiotic Resistance Genes in Two Different Wastewater Treatment Plant Systems in Poland: A Preliminary Study.

Antibiotic resistance is a growing problem worldwide. The emergence and rapid spread of antibiotic resistance determinants have led to an increasing concern about the potential environmental and public health endangering. Wastewater treatment plants (WWTPs) play an important role in this phenomenon since antibacterial drugs introduced into wastewater can exert a selection pressure on antibiotic-resistant bacteria (ARB) and antibiotic resistance genes (ARGs). Therefore, WWTPs are perceived as the main sources of antibiotics, ARB and ARG spread in various environmental components. Furthermore, technological processes used in WWTPs and its exploitation conditions may influence the effectiveness of antibiotic resistance determinants' elimination. The main aim of the present study was to compare the occurrence of selected tetracycline and sulfonamide resistance genes in raw influent and final effluent samples from two WWTPs different in terms of size and applied biological wastewater treatment processes (conventional activated sludge (AS)-based and combining a conventional AS-based method with constructed wetlands (CWs)). All 13 selected ARGs were detected in raw influent and final effluent samples from both WWTPs. Significant ARG enrichment, especially for tet(B, K, L, O) and sulIII genes, was observed in conventional WWTP. The obtained data did not show a clear trend in seasonal fluctuations in the abundance of selected resistance genes in wastewaters.

Genetic variation in IL-10 influences the progression of hepatitis B infection.

OBJECTIVES: The outcomes of hepatitis B virus (HBV) infection vary substantially among affected individuals, providing evidence of the role of host genetic background in the susceptibility to HBV persistence and the dynamics of liver injury progression to cirrhosis and hepatocellular carcinoma (HCC). METHODS: Six single-nucleotide polymorphisms within the interleukin 10 gene (IL10) were genotyped by MALDI-TOF mass spectrometry in 857 patients with chronic HBV infection (CHB), 48 patients with resolved HBV infection, and 100 healthy volunteers. Associations of the selected polymorphisms with susceptibility to chronic HBV infection, liver injury progression, and outcomes were investigated. RESULTS: IL10 -819T (rs1800871), -592A (rs1800872), and +504T (rs3024490) alleles were associated with treatment-induced hepatitis B surface antigen (HBsAg) seroclearance. Additionally, IL10 ATAC haplotype increased the chance of HBsAg loss and was significantly more frequent in patients with less liver injury. Moreover rs1800871TT, rs1518110TT, rs1800872AA, and rs3024490TT genotypes were identified as predictors of a lower FIB-4 score (<0.5). CONCLUSIONS: This study indicates that polymorphisms within the promoter region and intronic sequences of IL10 are associated with chronicity of hepatitis B and with HBV-induced liver damage.

Host genetic background affects the course of infection and treatment response in patients with chronic hepatitis B.

BACKGROUND: Hepatitis B virus (HBV) utilizes proteins encoded by the host to infect hepatocytes and replicate. Recently, several novel host factors have been identified and described as important to the HBV lifecycle. The influence of host genetic background on chronic hepatitis B (CHB) pathogenesis is still poorly understood. OBJECTIVES: Here, we aimed to investigate the association of NTCP, FXRα, HNF1α, HNF4α, and TDP2 genetic polymorphisms with the natural course of CHB and antiviral treatment response. STUDY DESIGN: We genotyped 18 single-nucleotide polymorphisms using MALDI-TOF mass spectrometry in 136 patients with CHB and 100 healthy individuals. We investigated associations of the selected polymorphisms with biochemical, serological and hepatic markers of disease progression and treatment response. RESULTS: No significant differences in genotypic or allelic distribution between CHB and control groups were observed. Within TDP2, rs3087943 variations were associated with treatment response, and rs1047782 modified the risk of advanced liver inflammation. Rs7154439 within NTCP was associated with HBeAg seroconversion after 48 weeks of nucleos(t)ide analogue treatment. HNF1α genotypes were associated with treatment response, liver damage and baseline HBeAg presence. HNF4α rs1800961 predicted PEG-IFNα treatment-induced HBsAg clearance in long-term follow up. CONCLUSIONS: This study indicates host genetic background relevance in the course of CHB and confirms the role of recently described genes for HBV infection. The obtained results might serve as a starting point for validation studies on the clinical application of selected genetic variants to predict individual risks of CHB-induced liver failure and treatment response.

TNF-α polymorphisms affect persistence and progression of HBV infection.

BACKGROUND: Hepatitis B virus (HBV) infections are a major threat worldwide. Disease progression and outcome is diverse and depends on host genetic background. Recently, a high rate of HBV reactivation in individuals receiving tumor necrosis factor-α (TNF-α) antagonists showed the importance of this cytokine in HBV infection control. Here, we investigated the influence of TNF-α promoter polymorphisms on susceptibility to chronic HBV infection (CHB), liver injury progression and outcomes. METHODS: A total of 231 patients with CHB constituted the study group and 100 healthy volunteers-the local control group. TNF-α -1031T/C, -863C/A, -857C/T, -308G/A, and -238G/A were genotyped using MALDI-TOF mass spectrometry. RESULTS: TNF-α -1031C and -863A alleles were observed more frequently in CHB group than in healthy controls. Carriers of TNF-α -1031C and -863A variant alleles had lower baseline levels of serum HBV DNA and lower liver necroinflammatory activity than dominant homozygotes. A -857CT genotype predisposed to higher necroinflammatory activity. No associations between TNF-α variants and liver fibrosis were found. CONCLUSION: This study indicates that TNF-α -863A and -1031C alleles are associated with increased susceptibility to CHB in individuals from northern Poland. The same variants determine the course of CHB, lowering viremia and reducing necroinflammatory activity of the liver.

Associations of ESR1 and ESR2 gene polymorphisms with metabolic syndrome and its components in postmenopausal women.

OBJECTIVES: Metabolic syndrome (MS) affects a quarter of Polish people and is associated with diabetes mellitus type 2 and ischemic heart disease. The prevalence of MS in postmenopausal women can be increased by the lack of protective effects of oestrogens. In the near future, because of the general increase in life expectancy, the number of postmenopausal women will rise substantially. Therefore, investigating both the environmental and the genetic factors predisposing to MS may have a great impact on women's health. The aim of this study was to determine whether particular oestrogen receptor (ESR) gene polymorphisms can predispose to the development of MS in women after menopause. STUDY DESIGN: The sample consisted of 147 postmenopausal women. In addition to collecting medical history and analyzing body composition using the TANITA scale, patient's waist size, blood pressure, serum lipids, glucose, insulin, C-reactive protein and adiponectin were measured. The analysis of ESR gene polymorphisms was performed using the Sequenom MassARRAY platform. RESULTS: Three out of ten analyzed polymorphisms in the ESR1 gene (rs2234693, rs6902771, rs7774230) and one out of eight analyzed polymorphisms in the ESR2 gene (rs3020449) were associated with MS. The ESR1 rs2234693, rs6902771 and rs7774230 polymorphisms were associated with serum concentrations of high-density lipoproteins. The ESR2 rs3020449 polymorphism was associated with serum concentrations of total cholesterol and low-density lipoprotein. Four ESR1 polymorphisms (rs1709183, rs2234693, rs6902771, rs7774230) were associated with total fat tissue content. CONCLUSIONS: Bearing the particular alleles at the ESR gene polymorphisms may impact the development of MS and some of the ESR polymorphisms may influence serum cholesterol concentrations in women after menopause.

Differences in sequences between HBV-relaxed circular DNA and covalently closed circular DNA.

The hepatitis B virus (HBV) genome exists in two forms: circular covalently closed DNA (cccDNA) and relaxed circular DNA (RCDNA). Here, we investigated the presence of differences in the sequences of both forms in paired samples of serum and liver tissue. The serum and liver biopsy samples were collected at the same time from 67 chronically infected patients. The genotyping of the RCDNA and cccDNA was performed using mass spectrometry analysis. The HBV mutations located in the HBV pol (P) and the HBV basal core promoter/pre-core (BCP/PC) regions were included. The BCP/PC and P sequences of the RCDNA extracted from liver and blood samples were different in 39% and 16% of patients, respectively. Differences were also found between RCDNA and cccDNA extracted from the same liver specimen. Moreover, the cccDNA BCP/PC region sequence had an impact on various virological and clinical parameters. We demonstrated that there are differences between the RCDNA and cccDNA sequences that were extracted from the same liver tissue. However, further investigations are needed to analyze whether the mutations in the cccDNA are conserved and whether cccDNA serves as a 'mutation storage' pool for HBV. This result could have profound implications for the subsequent therapy choices for treatment-experienced patients.

Analysis of polymorphism and hepatic expression of duodenal cytochrome b in chronic hepatitis C.

BACKGROUND AND AIM: Pathological iron overload is commonly found in chronic hepatitis C (CHC) patients and considered as a negative prognostic factor of the disease. A single nucleotide polymorphism (SNP) rs884409 in duodenal cytochrome b gene (CYBRD1) is implicated in the pathogenesis of hemochromatosis. In our study we investigated the impact of the CYBRD1 genotype and expression on iron overload in CHC patients. METHODS: Liver biopsy specimens and whole blood samples from 243 patients with CHC were included in the study. Iron deposits in hepatocytes, serum markers of iron overload, and expression profile of gene-regulators of iron homeostasis were analyzed. Genotyping and analysis of gene expression of the CYBRD1 were performed. The frequency of SNP and the expression levels of CYBRD1 were compared between the groups of patients with and without markers of iron overload. RESULTS: The single nucleotide variant rs884409 G was associated with elevated serum iron levels, increased markers of liver inflammation, and oxidative stress. Hepatic expression of CYBRD1 was associated with the expression of Tfr2, Id1, and HO-1 genes, serum ferritin levels, and with increased iron accumulation in liver. CONCLUSION: These results implicate CYBRD1 involvement in iron homeostasis in CHC.

Current molecular methods for the detection of hepatitis B virus quasispecies.

Chronic HBV infection affects more than 240 million people worldwide and is associated with a broad range of clinical manifestations including liver cirrhosis, liver failure and hepatocellular carcinoma. Because of the lack of an efficient cure for chronic hepatitis B, the main goal of antiviral therapy is the prevention of liver disease progression coupled with prolonged survival of patients. Because HBV viral load has been shown to be a crucial determinant of the progression of liver damage, these goals can be achieved as long as HBV replication can be suppressed. Unfortunately, long-term therapy with the low-to-moderate genetic barrier drugs, which are still recommended in a majority of developing countries, are strongly associated with HBV resistance development and treatment failure. In such cases, the precise and accurate determination of drug-resistant variants in an individual patient before treatment is important for a proper choice of first-line potent therapy. Nowadays, a number of techniques are available to study HBV quasispecies evolution. This review describes the advantages and limitations of various assays detecting drug-resistant HBV variants. Copyright © 2016 John Wiley & Sons, Ltd.

An initial assessment of correlations between host- and virus-related factors affecting analogues antiviral therapy in HBV chronically infected patients.

BACKGROUND: Success in treating hepatitis B virus (HBV) infection with nucleoside analogues drugs is limited by the emergence of drug-resistant viral strains upon prolonged therapy. In addition to mutation patterns in the viral polymerase gene, host factors are assumed to contribute to failure of treatment in chronic HBV infections. The aim of this study was to analyze the correlation between efficacy of antiviral therapy and the prevalence of HBV pretreatment drug-resistant variants. We also analyzed the role of heterogeneity in the promoter region of the IL-10 on the HBV pol/s gene polymorphisms and efficacy of analogues-driven therapy. MATERIAL AND METHODS: HBV DNA was extracted from 54 serum samples from chronic hepatitis B (CHB) patients. Drug-resistance mutations were analyzed using MALDI-TOF mass spectrometry technology (MALDI-TOF MS) and Multi-temperature single-strand conformation polymorphism (MSSCP). IL-10 gene promoter region polymorphisms at positions -1082, -819, and -592 were determined in allele-specific PCR reactions (AS-PCR). RESULTS: Drug-resistance mutations were detected in 74% of naïve and 93% of experienced patients, but the effect of pre-existence of drug-resistant HBV variants on antiviral therapy was not statistically significant (p=0.86). The role of polymorphisms at positions -1082 (p=0.88), -819 (p=0.26), and -592 (p=0.26) of IL-10 promoter region polymorphisms was excluded from the response-predicting factors. The main host factors predicting successful response to antiviral therapy were female sex (p=0.007) and young age (p=0.013). CONCLUSIONS: The presence of drug-resistant HBV variants in baseline is not a viral predictor of good response to nucleoside/nucleotide analogues therapy. Only low HBV viral load predicted positive response to antiviral therapy. The ideal candidate for antiviral therapy is an immunocompetent, young female with low HBV viral load and elevated ALT activity.

High-throughput matrix-assisted laser desorption ionization-time of flight mass spectrometry as an alternative approach to monitoring drug resistance of hepatitis B virus.

Long-term antiviral therapy of chronic hepatitis B virus (HBV) infection can lead to the selection of drug-resistant HBV variants and treatment failure. Moreover, these HBV strains are possibly present in treatment-naive patients. Currently available assays for the detection of HBV drug resistance can identify mutants that constitute ≥5% of the viral population. Furthermore, drug-resistant HBV variants can be detected when a viral load is >10(4) copies/ml (1,718 IU/ml). The aim of this study was to compare matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) and multitemperature single-strand conformation polymorphism (MSSCP) with commercially available assays for the detection of drug-resistant HBV strains. HBV DNA was extracted from 87 serum samples acquired from 45 chronic hepatitis B (CHB) patients. The 37 selected HBV variants were analyzed in 4 separate primer extension reactions on the MALDI-TOF MS. Moreover, MSSCP for identifying drug-resistant HBV YMDD variants was developed and turned out to be more sensitive than INNOLiPA HBV DR and direct sequencing. MALDI-TOF MS had the capability to detect mutant strains within a mixed viral population occurring with an allelic frequency of approximately 1% (with a specific value of ≥10(2) copies/ml, also expressed as ≥17.18 IU/ml). In our study, MSSCP detected 98% of the HBV YMDD variants among strains detected by the MALDI-TOF MS assay. The routine tests revealed results of 40% and 11%, respectively, for INNOLiPA and direct sequencing. The commonly available HBV tests are less sensitive than MALDI-TOF MS in the detection of HBV-resistant variants, including quasispecies.