RT Region Research Articles

The Characteristic of HBV Quasispecies Is Related to Occult HBV Infection of Infants Born to Highly Viremic Mothers.

Although a combination of immunoprophylaxis and antiviral therapy can effectively prevent mother-to-child transmission (MTCT) of hepatitis B virus (HBV), a considerable number of infants born to highly viremic mothers still develop occult HBV infection (OBI). To uncover the virological factor and risk predictor for OBI in infants, we found that the diversity and complexity of maternal HBV quasispecies in the case group were lower than those in the control group. Mutations with significant differences between the two groups were most enriched in the NTCPbd and PreC regions. Genetic distance at the amino-acid level of the PreC region, especially the combination of three amino-acid mutations in the PreC region, could strongly predict the risk of OBI in infants. HBV quasispecies in OBI infants were highly complex, and the non-synonymous substitutions were mainly found in the RT and HBsAg regions. The sK47E (rtQ55R) and sP49L mutations in OBI infants might contribute to OBI through inhibiting the production of HBV DNA and HBsAg, respectively. This study found the potential virological factors and risk predictors for OBI in infants born to highly viremic mothers, which might be helpful for controlling OBI in infants.

Prevalence of occult hepatitis B infection among blood donors in Saint Petersburg

The aim of this study was to assess the prevalence of occult hepatitis B infection among blood donors in St. Petersburg, as well as to characterize the identified virus isolates. The study material was represented by 2800 blood plasma samples collected in 2019 from blood donors living in St. Petersburg. The ELISA study for HBV marker rate consisted of HBsAg, anti-HBs IgG, anti-HBcore IgG. HBV DNA was analyzed by nested PCR with real-time hybridization-fluorescence detection on three targets allowing to determine virus DNA at low viral load, including HBsAg-negative chronic hepatitis B. Hepatitis B serological markers were detected in 69.43% of those surveyed, HBsAg was found in 0.43% of individuals, and all of which donated blood first time. A significant excess of the anti-HBcore IgG antibodies occurrence among primary donors (15.1%) compared with repeated/regular donors (7.48%) was shown. The prevalence of virus DNA in the group was 3.14%, including 2.71% of cases in HBsAg-negative CHB. Based on phylogenetic analysis of 88 isolates, HBV subgenotypes were determined in the following order: D1 and D2, 40.91% each, D3 and A2, 9.09% each. While determining the serological subtype in detected isolates, the serotype ayw3 (52.27%) vs ayw2 (46.59%) and adw2 (10.23%) prevailed. Drug resistance mutations, including compensatory ones, were detected in six examined patients (6.82%). In all genotype D isolates, multiple amino acid substitutions were identified in the RT, SHB, MHB, LHB, and Core regions; mutations in the preCore region were detected in 21.59% samples. In the MHR of the HBV genotype D genome, twenty-six positions were identified in which amino acid substitutions occurred, and all isolates showed modifications at positions 113, 114, 131, 134, 159, 161, 168, in 76 — at position 122, in 68 — at position 127, in 36 — at position 118, in 24 — at position 128. In HBV A2 isolates, mutations T113S, S143T, Y161F were identified. Nine isolates in the preCore region showed a polymorphism including a stop codon W28*W; in five isolates the W28S substitution was shown in the same position, and the W28*S variant was found in one more sample. The high incidence of HBsAg-negative CHB cases among blood donors, as well as the predominance of HBV isolates that simultaneously carry mutations resulting in diagnostic failure of HBsAg tests and prophylactic failure of immunoglobulin or vaccines and virus reactivation, mutations that contribute to disease progression obviously pose a threat to health and require to be further examined.

Molecular Investigation of Occult Hepatitis B Virus Infection Among Patients with Liver Cancers

Background: Hepatitis B virus (HBV) is still the leading cause of hepatocellular carcinoma (HCC). HBV could persist in the low replicate state that is defines as occult hepatitis B virus infection (OBI). Recently, OBI has been defined important in the development and/or exacerbation of HCC and other liver diseases. Objectives: This study tried to determine the frequency of OBI among liver tumor samples. Methods: This cross-sectional study was performed among patients with HCC and intrahepatic cholangiocarcinoma (iCCA) in hospitals affiliated with Iran University of Medical Sciences. Liver tumor samples (Fresh frozen and formalin fixed paraffin embedded) were processed for isolation of DNA and then subjected to molecular assays, including PCR examinations of HBV genes (S, X, and C), determination of viral loads, detection of HBV-cccDNA, phylogenetic analysis, and assessment of mutations in HBsAg and RT regions. Results: In total, there were 93 participants, including HCC (n = 60), iCCA (n = 33); among which, 15% were detected positive for OBI. The OBI among HCC and iCCA were 18.3% and 9.1%, respectively. The mean intrahepatic viral load was 1.3 × 105 ± 1.4 × 102 copies/µl, and 5 had detectable cccDNA. The OBI subjects belonged to the HBV genotype D and subgenotype D1. In the HBsAg protein, T45N (33.3%) and P105A (25%) were the most prevalent mutations. N53K (33.3%), Y54H (25%), L80I (33.3%), and A113G (25%) were missense mutations that were observed in the RT domain. Conclusions: HBV-DNA was detected among liver tumor samples with negative HBsAg status. The results of viral load and cccDNA tests are important in identification and prognosis of liver diseases. It’s needed more precise molecular and cohort studies to clarify the functions of OBI in liver diseases.

Analysis of characteristics of drug resistance gene mutation in HBV RT region of hepatitis B infected patients

Objective: This article investigated the clinical characteristics and distribution of drug resistance mutation sites in HBV RT region of hepatitis B infected patients. Methods: Retrospective analysis was made on 1 948 patients with HBV infection, who had been tested for NAs resistance mutation and had a medical history of NAs in the Laboratory Department of the Fifth Medical Center of the PLA General Hospital from January 2020 to December 2021. Basic clinical information and drug resistance related mutation information were recorded. Meanwhile, the serological index data of hepatitis B were collected. Drug resistance gene mutant group and non-mutated group were grouped according to whether the drug resistance genes had a mutation in HBV RT region, and the clinical characteristics and genotype distribution of the two groups were statistically analyzed. The pattern of drug resistance gene mutation, number of mutation sites, drug resistance type and mutation of NAs resistance-related sites were analyzed in 917 patients with drug resistance gene mutation in HBV RT region. χ2 Inspection was used for counting data. Meanwhile, two independent samples t-test and Wilcoxon rank sum test were used for measurement data. Results: Among the 1 948 patients with chronic HBV infection, 917 patients had drug resistance gene mutation in RT region (47.07%). The proportion of patients with acute hepatitis B and CHB in HBV RT resistance gene mutant group was lower than that in the non-mutated group, while the proportion of patients with HBV-related cirrhosis was higher than that in the non-mutated group, these differences were statistically significant. Compared with the non-mutated group in HBV RT region, the age, the positive rates of HBeAg and HBV DNA, and HBV DNA load of these patients were increased in drug resistance gene mutant group, these differences were statistically significant. Genotypes of patients in both groups were dominated by C, followed by B and D. The proportion of patients with genotype C in HBV RT drug resistance gene mutant group was higher than that of non-mutated group, the difference was statistically significant. There were 53 gene mutation patterns in 917 patients with drug resistance gene mutation in HBV RT region, and the main pattern was rtL180M+rtM204V+rtS202G (9.70%). The mutation sites were dominated by 3 (20.74%). There were 5 types of drug resistance, LAM+Ldt (21.25%) was the most. Among the 18 sites that were clearly associated with LAM, ADV, ETV and Ldt resistance in the HBV RT region, 14 sites were mutated, and the most common mutation sites were rtL180M, rtM204V, rtM204 and rtS202G. what's more, the proportion of patients with NAs drug resistance was LAM>Ldt>ETV>ADV. Conclusion: In order to prevent adverse consequences of this study such as disease recurrence or disease progression caused by HBV drug resistance, HBV infected patients, who have long-term use of NAs antiviral therapy, should monitor the level of HBV DNA and drug resistance genes in HBV RT region in order to optimize the treatment plan in time or guide individualized treatment.

A Concentration Method for HIV Drug Resistance Testing in Low-Level Viremia Samples.

Drug resistance testing in HIV-1 low-level viremia (LLV) samples is challenging yet critical. Our study is aimed at assessing the performance of lentivirus concentration reagent (LCR) in combination with a validated Sanger sequencing (SS) for monitoring drug resistance mutations (DRMs) in LLV samples. A series of clinical samples were diluted and amplified for genotypic resistance testing (GRT) to prove the performance of the LCR. The Stanford HIV-1 drug resistance database (HIVdb version 8.9) was used to analyze the mutations. HIV-1 subtypes and CRFs were determined using the COMET online tool. The overall success rate of genotyping was compared with ultracentrifugation combined with SS. Furthermore, the success rates at varied VL of the two concentration methods were evaluated, and the DRMs of diluted samples were compared with those undiluted samples. When LCR was used, the overall success rate was 90% (72/80) in the PR and RT regions and 60% (48/80) in the IN region. In addition, when HIV RNA was 1000 copies/ml, 400 copies/ml, 200 copies/ml, and 100 copies/ml, the success rates of PR and RT regions were 100%, 100%, 95%, and 65%, respectively, while the success rates of IN region were 85%, 60%, 45%, and 50%, respectively. We found that the sample DR-387A2 missed the E138A mutation, and mutations in other samples were consistent with undiluted samples using LCR. LCR will support monitoring DRMs in HIV-1 patients with LLV and can be an effective alternative for small- and medium-sized laboratories that cannot afford an ultracentrifuge.

Whole genome analysis of hepatitis B virus before and during long-term therapy in chronic infected patients: Molecular characterization, impact on treatment and liver disease progression.

Hepatitis B virus (HBV) infection remains a serious public health concern worldwide despite the availability of an efficient vaccine and the major improvements in antiviral treatments. The aim of the present study is to analyze the mutational profile of the HBV whole genome in ETV non-responder chronic HBV patients, in order to investigate antiviral drug resistance, immune escape, and liver disease progression to Liver Cirrhosis (LC) or Hepatocellular Carcinoma (HCC). Blood samples were collected from five chronic hepatitis B patients. For each patient, two plasma samples were collected, before and during the treatment. Whole genome sequencing was performed using Sanger technology. Phylogenetic analysis comparing the studied sequences with reference ones was used for genotyping. The mutational profile was analyzed by comparison with the reference sequence M32138. Genotyping showed that the studied strains belong to subgenotypes D1, D7, and D8. The mutational analysis showed high genetic variability. In the RT region of the polymerase gene, 28 amino acid (aa) mutations were detected. The most significant mutations were the pattern rtL180M + rtS202G + rtM204V, which confer treatment resistance. In the S gene, 35 mutations were detected namely sP120T, sT126S, sG130R, sY134F, sS193L, sI195M, and sL216stop were previously described to lead to vaccine, immunotherapy, and/or diagnosis escape. In the C gene, 34 mutations were found. In particular, cG1764A, cC1766G/T, cT1768A, and cC1773T in the BCP; cG1896A and cG1899A in the precore region and cT12S, cE64D, cA80T, and cP130Q in the core region were associated with disease progression to LC and/or HCC. Other mutations were associated with viral replication increase including cT1753V, cG1764A/T, cC1766G/T, cT1768A, and cC1788G in the BCP as well as cG1896A and cG1899A in the precore region. In the X gene, 30 aa substitutions were detected, of which substitutions xT36D, xP46S, xA47T, xI88F, xA102V, xI127T, xK130M, xV131I, and xF132Y were previously described to lead to LC and/or HCC disease progression. In conclusion, our results show high genetic variability in the long-term treatment of chronic HBV patients causing several effects. This could contribute to guiding national efforts to optimize relevant HBV treatment management in order to achieve the global hepatitis elimination goal by 2030.

Effect of mutations across reverse transcriptase region on HBV replication and progression of liver diseases in Chinese patients.

It was known that mutations in the RT region were mainly related to nucleot(s)ide analogs resistance. Increasing studies indicated that RT mutations were related to advanced liver diseases (ALD) and had effects on HBV replication, but the distribution characteristics of mutations across RT region in the development of liver diseases and the effect of RT mutations on HBV replication were not fully clarified. HBV RT region was direct‐sequenced in 1473 chronic HBV‐infected patients. Mutation frequencies were analyzed to identify the specific mutations differing between groups classified by genotypes, loads of HBV DNA, or progression of liver diseases. In the range of rt145‐rt290, rt145, rt221, rt222, rt267, and rt271 were the genotype‐polymorphic sites, while rt238 was the genotype‐specific sites. Mutations at rt163, rt173, rt180, rt181, rt184, rt191, rt199, and rt214 were more frequent among patients with C‐genotype HBV, while those at rt220, rt225, rt226, rt269, and rt274 were more frequent among patients with B‐genotype HBV. RtM204V/I could reduce the HBV DNA loads while rtQ/L267H/R could increase the HBV DNA loads. RtV214A/E/I (OR 3.94, 95% CI 1.09 to 14.26) was an independent risk factor for advanced liver diseases. In summary, the hotspots of mutations were different between B and C genotypes. Besides the effect on the S region, RT mutations had effects on HBV replication by other unknown ways. RtV214A/E/I was found to be an independent risk factor for ALD, suggesting that mutations at rt214 site could be used as a potential virological marker for the liver disease progression.

The dynamic changes of HBV quasispecies diversity in infancy after immunoprophylaxis failure: a prospective cohort study

BackgroundPrevious works have observed that younger infants with chronic hepatitis B virus (HBV) infection are more responsive to antiviral treatment. However, the underlying mechanism remains unclear. In this study, the dynamic changes of HBV quasispecies in infants with immunoprophylaxis failure were investigated to provide virological explanations for clinical management on infantile antiviral therapy.MethodsThirteen 7-month-old infants with immunoprophylaxis failure and their mothers were enrolled from a prospective cohort, and 8 of them were followed up to 3 years old. The sequences of HBV quasispecies were analyzed by the full-length genome clone-based sequencing, and compared among mothers and their infants at different ages.ResultsThe results revealed that the complexity, mutation frequency and genetic distance of HBV quasispecies decreased significantly at full-length, partial open reading frames and regulatory regions of HBV genome at nucleotide level in 7-month-old infants comparing with their mothers, whereas increased significantly to near the maternal level when infants grew up to 3 years old. Furthermore, similar changes were also found in Core, PreS2, RT and P regions of HBV genome at amino acid level, especially for potential NAs-resistant mutants in RT region and immune-escape mutants in Core and PreS2 regions.ConclusionsThis study uncovered the evolution of HBV quasispecies in infancy after mother-to-child transmission, which may provide the virological evidence for explaning that younger children are more responsive to antiviral therapy.

Drug resistance profiles and influencing factors among HIV-infected children and adolescents receiving long-term ART: a multicentre observational study in China.

To analyse the characteristics and determinants of drug resistance mutations (DRMs) in HIV-infected children and adolescents on long-term ART in China. An observational cohort study was conducted in five centres. All participants younger than 15 years at ART initiation were screened, and those identified as having virological failure (VF) with viral load (VL) ≥ 400 copies/mL were included for genotypic resistance testing. Logistic regression analysis was performed and the accumulation of major mutations was analysed in a subgroup of resistant individuals with complete VL results since HIV diagnosis. Among 562 eligible participants, protease and RT regions were successfully amplified for 93 who failed treatment with a median of 10.0 years ART. Sixty-eight (73.1%) harboured ≥1 major mutations. NRTI, NNRTI and dual-class resistance accounted for 48.4%, 63.4% and 38.7%, respectively. Only 3.2% were resistant to PIs. Age at ART initiation [adjusted OR (aOR) = 0.813, 95% CI 0.690-0.957], subtype B (aOR = 4.378, 95% CI 1.414-13.560) and an initial NNRTI-based regimen (aOR = 3.331, 95% CI 1.180-9.402) were independently associated with DRMs. Among 40 resistant participants with additional VL data, 55.0% had continued VF on a suboptimal regimen and the estimated duration of VF was positively correlated with the total number of major mutations (r = 0.504, P = 0.001). The development of DRMs was common in children and adolescents receiving long-term treatment, and continued VF was prevalent in those with resistance. Timely genotypic testing and new child-friendly formulations are therefore urgently required.

Prevalence and patterns of HIV drug resistance in patients with suspected virological failure in North-Western Tanzania.

BackgroundMore than 15 million people in sub-Saharan Africa receive ART. Treatment failure is common, but the role of HIV drug resistance in treatment failure is largely unknown because drug resistance testing is not routinely done. This study determined the prevalence and patterns of HIV drug resistance in patients with suspected virological failure.Materials and methodsA single high viral load of >1000 viral RNA copies/mL of plasma at any point during ART was considered as suspected virological failure. HIV-1 RNA was extracted from plasma samples of these patients using the QIAamp Viral RNA kit. The protease and part of the RT regions of the HIV pol gene were characterized.ResultsViral load was determined in 317 patients; 64 (20.2%) had suspected virological failure. We successfully genotyped 56 samples; 48 (85.7%) had at least one major resistance-associated mutation (RAM). Common mutations in RT were M184V (75%), T215Y (41.1%), K103N (39.3%), M41L (32.1%), D67DN (30.3%), G190A (28.6%) and A98G (26.8%). No RAMs were detected in ART regimens based on a ritonavir-boosted PI.ConclusionsThe Tanzanian national guidelines define ‘virological failure’ as two consecutive viral load measurement results, at 3 month intervals, above the WHO threshold (1000 copies/mL). Here, we show that a single viral load above the WHO threshold is associated with high rates of RAMs. This suggests that a single high viral load measurement could be used to predict virological failure and avoid delays in switching patients from first-line to higher genetic barrier second-line regimens.

Analysis of risk factors for drug resistant mutations in the reverse transcriptase region in chronic hepatitis B virus-infected patients

Objective: To analyze the risk factors that may affect the mutations in the reverse transcriptase region in chronic hepatitis B virus-infected patients. Methods: 678 hospitalized cases with chronic HBV infection who underwent HBV RT testing at Tianjin Second People's Hospital from January 1, 2016 to December 31, 2016 were collected retrospectively. Among them, 417 cases were diagnosed with chronic hepatitis B, 219 cases with liver cirrhosis and 42 cases with primary liver cancer. There were 268 cases of non-use of any antiviral therapy, 138 cases of discontinuation of antiviral drugs for 6 months or more, and 272 cases of continuous antiviral therapy. HBV genotyping and RT region mutation sites were detected by direct sequencing. The risk factors that may affect the drug resistant mutation in the HBV RT mutation, including age, genotype, antiviral drug selection and medication time, hepatitis B virus infection, and biochemical markers were analyzed by univariate analysis to screen out independent risk factors. Results: Among 678 HBV-infected cases, 290 cases (42.8%) were detected with RT-region mutation. Among them, the pre-existing drug resistant rate was 6.72%, and the drug resistant mutation rate was 23.19% in treated patients. The drug resistant mutation rate of patients with continuous antiviral therapy was 66.18%. Gene mutations highest rate for 1 ~ 5 years was 27.14% in chronic HBV patients treated with antiviral therapy. Logistic regression analysis of the factors that had led to HBV mutation showed that old age, the selection of nucleoside drugs at the beginning of treatment and medication time were the main factors affecting HBV RT mutations. Conclusion: Abnormal ALT level, HBV genotype, HBV DNA quantitative level are the main factors influencing non-drug resistant mutations. Age over 60 years old, and long-term use of low-barrier nucleoside drugs are high-risk groups for HBV resistant. Therefore, HBV resistant monitoring should be strengthened.

A novel trigger-substrate mechanism based on clinically concealed repolarization abnormalities underlies idiopathic ventricular fibrillation

Abstract Background Sudden cardiac arrest (SCA) is most often due to ventricular fibrillation (VF). When no cause is found during diagnostic follow-up, fibrillation is classified as idiopathic (iVF). We hypothesize that a critical functional substrate-trigger interaction underlies iVF. Purpose To study electrophysiological triggers and substrate for iVF in a clinical cohort; and seek mechanistic explanations in explanted pig hearts and computer models mimicking trigger-substrate interactions. Methods Repolarization time (RT) isochrones on the epicardium were studied with electrocardiographic imaging (ECGI) in patients with iVF, patients with frequent monomorphic premature ventricular complexes (fmPVC) but no structural disease or SCA, and controls without cardiovascular disease. RT gradients were created in explanted, Langendorff-perfused pig hearts by local infusion of dofetilide (“dof”, 250 nM, delaying RT) and pinacidil (“pin”, 30 μM, shortening RT) in adjacent regions of the heart. Arrhythmia inducibility was tested by programmed stimulation (8 atrial stimuli [S1] followed by one ventricular stimulus [S2] paced at regions of early or late RT). A computational ventricular monodomain model was used to study the location-dependency of trigger-substrate interaction; RT gradients were created by local changes in potassium channel conductance. Results Although QTc values were similar, iVF survivors (n=11) displayed significantly steeper RT gradients than controls (n=10) or fmPVC individuals (n=7): 269±111 vs 179±40 vs 171±76 ms/cm respectively (panel A). Unipolar electrograms (EGMs) at the gradients displayed a change in polarity of the local T wave (B). In iVF, PVCs originated more often from regions with early RT than in fmPVC individuals (yellow circles in A; 64% vs 14%). In the explanted hearts (C), drug infusion resulted in similar RT gradients and polarity changes of EGM T waves (D-E). VF inducibility by pacing of the early RT region (D) increased significantly with steeper RT gradients (baseline: 3/6 hearts inducible, dof+pin: 3/3). Pacing of late RT regions (E) did not induce arrhythmias in baseline (0/6) nor with RT gradients (0/3). For similar pacing intervals at the early RT region, the 12-lead ECG R-on-T morphology was similar but VF only occurred in the presence of RT gradients (F). In the computer model, the number of inducible pacing intervals critically depended on the stimulus location (G). Conclusion Combined, these results demonstrate that R-on-T superposition per se is insufficient to explain arrhythmogenesis. Rather, not only the temporal coupling interval but also the spatial origin of PVCs in relationship to the degree of local repolarization abnormalities are critical elements. In iVF, a substrate of RT gradients (panel H1) with triggers from early RT regions (H2) precipitate reentry (H3). Noninvasive ECGI can uncover these substrate and trigger characteristics in (at least a subset of) iVF survivors. Funding Acknowledgement Type of funding source: Public grant(s) – National budget only. Main funding source(s): Netherlands Organization for Scientific Research Veni grant TTW 16772, French National Research Agency (ANR-10-IAHU04-LIRYC)

Genetic Diversity and Drug Resistance of HIV-1 CRF55_01B in Guangdong, China.

HIV-1 CRF55_01B was first reported in 2013. At present, no report is available regarding this new clade's polymorphisms in its functionally critical regions protease and reverse transcriptase. To identify the diversity difference in protease and reverse transcriptase between CRF55_01B and its parental clades CRF01_AE and subtype B; and to investigate CRF55_01B's drug resistance mutations associated with the protease inhibition and reverse transcriptase inhibition. HIV-1 RNA was extracted from plasma derived from a MSM population. The reverse transcription and nested PCR amplification were performed following our in-house PCR procedure. Genotyping and drug resistant-associated mutations and polymorphisms were identified based on polygenetic analyses and the usage of the HIV Drug Resistance Database, respectively. A total of 9.24 % of the identified CRF55_01B sequences bear the primary drug resistance. CRF55_01B contains polymorphisms I13I/V, G16E and E35D that differ from those in CRF01_AE. Among the 11 polymorphisms in the RT region, seven were statistically different from CRF01_AE's. Another three polymorphisms, R211K (98.3%), F214L (98.3%), and V245A/E (98.3 %.), were identified in the RT region and they all were statistically different with that of the subtype B. The V179E/D mutation, responsible for 100% potential low-level drug resistance, was found in all CRF55_01B sequences. Lastly, the phylogenetic analyses demonstrated 18 distinct clusters that account for 35% of the samples. CRF55_01B's pol has different genetic diversity comparing to its counterpart in CRF55_01B's parental clades. CRF55_01B has a high primary drug resistance presence and the V179E/D mutation may confer more vulnerability to drug resistance.

Evidence of genomic information and structural restrictions of HIV-1 PR and RT gene regions from individuals experiencing antiretroviral virologic failure

ObjectivesThis study analyzed Protease-PR and Reverse Transcriptase-RT HIV-1 genomic information entropy metrics among patients under antiretroviral virologic failure, according to the numbers of virologic failures or resistance mutations. MethodsFor this purpose, we used genomic sequences from PR and RT of HIV-1 from a cohort of chronic patients followed up at São Paulo Hospital. ResultsInformational entropy proportionally increases with the number of antiretroviral virologic failures in PR and RT (p < .001). Affected regions of PR were related to catalytic and structural functions, such as Fulcrum (K20) Flap (M46) and Cantilever (A71). In RT, this occurred at Fingers (E44) and Palm (K219). Informational entropy increases according to the number of resistance mutations in PR and RT (p < .001). Higher PR entropy was proportional to the resistance mutation numbers in Fulcrum (L10), Active site (L24) Flap (M46), Cantilever (L63) and near Interface (L90). In RT, they related to regions responsible for protein stability such as Fingers (T39) and Palm (L100). ConclusionsThe antiretroviral selective pressure affects HIV genomic informational entropy at the PR and RT regions, leading to the emergence of more unstable virions. Mapping the three-dimensional structure in these HIV-1 proteins is relevant to designing new antiretroviral targeting resistant strains.

Mutations in reverse transcriptase region of HBV affect Hepatitis B surface antigen titers and its correlation with HBV DNA.

The impact of mutations in the reverse transcriptase region of HBV on serum HBsAg titer and its correlation with HBV DNA is largely unknown. A total of 644 patients, with a history of lamivudine or adefovir dipivoxil resistance who underwent genotypic resistance tests, were enrolled in this study. Serum HBsAg, hepatitis B e antigen and HBV DNA were quantified, and the HBV RT region was sequenced and analyzed. Then, the patients were divided into five sub-groups, including M204I/V, L180M+M204I/V, A181T/V, N236T and A181T/V+N236T according to the mutation spectra. HBsAg was lower in the wild-type and A181T/V+N236T groups as compared to the M204I/V, L180M+M204I/V and N236T groups. HBsAg was positively correlated with HBV DNA levels in the wild-type group (r = 0.322, p < 0.01), as well as in the M204I/V, L180M+M204I/V, A181T/V, and N236T subgroups, while no correlation was found in the A181T/V+N236T subgroup (r = 0.159, p = 0.217). Moreover, for patients with N236T mutation, HBsAg was positively correlated with HBV DNA level in the HBeAg negative group (r = 0.435, p = 0.016), but not in the HBeAg positive group (r = 0.105, p = 0.594). For patients with A181T/V or N236T mutation, HBsAg was positively correlated with HBV DNA in older patients (≥ 40 years), but not in younger patients (< 40 years). Serum HBsAg titer and its correlation with HBV DNA may be affected by mutations in the reverse transcriptase region of HBV, that should be re-evaluated in patients with antiviral resistance.

Molecular Epidemiology of Hepatitis B Virus in Turkish Cypriot.

There is an increased demand for molecular and epidemiological information regarding Hepatitis B Virus (HBV) infection as the disease severity depends on these specifications. We have aimed to analyze nucleos(t)ide analogues (NA) resistance and typical HBsAg escape mutations with the dispersion of HBV genotype/subgenotype/HBsAg serotypes on overlapping pol/S gene regions in the Turkish population. Samples were collected in Northern Cyprus. Reverse transcriptase (rt) region between 80–250 amino acids were amplified. Typical HBsAg escape mutations were determined as HBIg escape (6.48%), vaccine escape (8.34%), HBsAg misdiagnosis (9.25%), and immune escape mutations (8.34%). NAs resistances were determined as primary (2.78%), partial (2.78%), and compensatory mutations (26.85%) in overlapping pol/S gene region. The study patients were predominantly infected with HBV genotype D/D1 (98%). However, the predominant HBsAg serotype was ayw2 (99%). The most common NA resistance mutation was rtQ215H/P/S (16.67%), however, for S gene the misdiagnosis mutations were observed most frequently (9.25%). We can conclude that HBV D/D1 is the dominant strain and ayw2 is the dominant serotype in the Turkish Cypriot. Cyprus is an island located in the Eastern Mediterranean region, and it is, therefore, a key location for human trafficking and immigration; as a result of this reputation, it is necessary to analyze HBV phylogenetically for local dynamics, and our results indicate that treatment naïve population is prone to these pol/S gene mutations. However, if HBV strains were also analyzed among Greek Cypriots too, this would enable a complete island survey. With this work, we believe that we have enlightened this subject for further research.

In silico study of pseudoprogression in glioblastoma: collaboration of radiologists and radiation oncologists in the estimation of extent of high dose RT region.

Oncologists play a vital role in the interpretation of radiographic results in glioblastoma patients. Molecular pathology and information on radiation treatment protocols among others are all important for accurate interpretation of radiology images. One important issue that may arise in interpreting such images is the phenomenon of tumor "pseudoprogression"; oncologists need to be able to distinguish this effect from true disease progression.Exact knowledge about the location of high-dose radiotherapy region is needed for valid determination of pseudoprogression according to RANO (Response Assessment in Neuro-Oncology) criteria in neurooncology. The aim of the present study was to evaluate the radiologists' understanding of a radiotherapy high-dose region in routine clinical practice since radiation oncologists do not always report 3-dimensional isodoses when ordering follow up imaging. Eight glioblastoma patients who underwent postresection radiotherapy were included in this study. Four radiologists worked with their pre-radiotherapy planning MR, however, they were blinded to RT target volumes which were defined by radiation oncologists according to current guidelines. The aim was to draw target volume for high dose RT fields (that is the region, where they would consider that there may be a pseudoprogression in future MRI scans). Many different indices describing structure differences were analyzed in comparison with original per-protocol RT target volumes. The median volume for RT high dose field was 277 ccm (range 218 to 401 ccm) as defined per protocol by radiation oncologist and 87 ccm (range 32-338) as defined by radiologists (median difference of paired difference 31%, range 15-112%). The Median Dice index of similarity was 0.46 (range 0.14 - 0.78), the median Hausdorff distance 25 mm. Continuing effort to improve education on specific procedures in RT and in radiology as well as automatic tools for exporting RT targets is needed in order to increase specificity and sensitivity in response evaluation.

1281. Emergence of a B/F1 HIV Recombinant in the Philippines: A Potentially New Circulating Recombinant Form

BackgroundThe Philippines has one of the fastest growing HIV epidemics globally. This was accompanied by a switch from subtype B to CRF01_AE. With a large population of returning overseas workers, new subtypes are being introduced regularly. Because diagnosis of HIV in the Philippines is usually late, superinfections can occur and may give rise to new circulating recombinant forms (CRFs). We propose a new CRF from the Philippines.MethodsFollowing institutional board approval, treatment-naive patients from two HIV treatment hubs (San Lazaro Hospital and the Philippine General Hospital) were recruited. Blood samples underwent Sanger sequencing of the PR and RT regions and next-generation sequencing (NGS) of near-full length genomes. Sequences were analyzed for recombination using the online tool jumping profile Hidden Markov Model (http://jphmm.gobics.de/).Results247 samples underwent Sanger sequencing of the PR and RT regions of the pol gene. Phylogenetic analysis indicated a clustering of four of the samples. Further analysis showed all four samples had the same breakpoints at nucleotides 2875, 2996, and 3001 (HXB2 numbering). All four patients were male, MSM, with a mean age of 28 years old (24–32), and >10 sexual partners. Mean CD4 count was 464 cells/µL and median viral load was 2.67 × 104 copies/mL. Two patients had sex with foreigners. To get a better overall view of subtype composition, we performed NGS using Illumina HiSeq. NGS showed the majority of the genome to be subtype F1 with segments of subtype B inserted in the pol, vpu, and env genes. A blast analysis of the consensus sequence showed 8,932 out of 8,943 nucleotides (99%) matched a 1999 sample from Argentina. Phylogenetic analysis of these samples show clustering of the four B/F1 recombinants with some South American sequences. No drug resistance mutations were identified.ConclusionMutation and recombination contribute to the extensive genetic diversity of HIV. Understanding this is important in choosing treatment regimens, developing future vaccines, and pursuing epidemiological investigations. The emergence of a new CRF in the Philippines underlies the importance of conducting routine surveillance for new HIV recombinant forms.Figure 1.New CRF genome showing subtype components.Disclosures All authors: No reported disclosures.

Predictors of Therapeutic Outcome to Nucleotide Reverse Transcriptase Inhibitor in Hepatitis B Patients.

Hepatitis B is a clinically important public health issue. Infection leads to hepatocellular carcinoma. Therefore, patients need antiviral therapy for prolonged period to prevent the complication of the disease. Data concerning chronic hepatitis B (CHB) patients with high hepatitis B virus (HBV) DNA are limited. The aim of the study was to check the efficacy of the nucleoside reverse transcriptase inhibitors (tenofovir) in terms of suppression of HBV DNA. The secondary end point in the study is to evaluate trends of predictive variables that predict outcome of treatment. In this specific study, we evaluated 140 CHB male and female patients, of these 110 completed 48 weeks of treatment. On the basis of hepatitis B e antigen (HBeAg), patients were stratified; HBV DNA and hepatitis B surface antigen (HBsAg) levels were measured along with liver function tests. All enrolled patients were given tenofovir disoproxil fumarate 300 mg daily before breakfast. Overall, 69.1% of patients showed virologic response. HBeAg-negative patient group showed 68% viral suppression and HBeAg-positive patient group showed 45.9% over 24 months of treatment, while at 48 months it was shown to be 76.7% and 54.1%, respectively. None of the patients suffered HBsAg loss during the 48 months. Baseline high HBV DNA level was found as a significant predictor of response (OR, 1.9; 95% CI = 1.23-3.9, p = 0.005). None of the patients observed had serious adverse events. Mutations in the RT region of polymerase gene are shown to be associated with resistance to antiviral drugs. Among patients suffering with chronic HBV infection, HBeAg-negative patient group have better virologic response as compared with HBeAg-positive group. Higher concentration of HBV DNA at baseline has negative prediction for sustained viral suppression. The A-B motif interdomain rtL122F mutation was found in nonresponder patients in our study. Another mutation rtN248H observed in E motif considered to have effect on DNA primer grip, which forms part of binding pocket.

P01.027 Pseudoprogression in glioblastoma: how sure are radiologists in location of “high-dose RT region”

P01.027 Pseudoprogression in glioblastoma: how sure are radiologists in location of “high-dose RT region”