Evaluation of the Abbott HBV RUO Sequencing Assay Combined with Laboratory-Modified Interpretive Software

The Abbott HBV RUO Sequencing assay (Abbott Molecular Inc., Des Plaines, IL), which combines automated sample processing, real-time PCR, and bidirectional DNA sequencing, was evaluated for detection of nucleos(t)ide analogue (NA) resistance-associated mutations located in the hepatitis B virus (HBV) polymerase (Pol) gene. Interpretive software from the assay manufacturer was modified to allow interrogation of the overlapping HBV surface (S) gene sequence for HBV genotype determination and detection of immune escape mutations. Analytical sensitivity (detection and sequencing) of the assay was determined to be 103.9 IU/ml (95% confidence interval [CI], 80.0 to 173.3) for HBV genotype A. Testing of commercially available HBV genotype panels consisting of 23 individual members yielded complete agreement between expected results and results obtained from the laboratory-developed HBV genotype library. Excellent specificity was observed among clinical specimens with serologic or molecular markers for various unrelated blood-borne viruses (n = 6) and sera obtained from healthy, HBV-negative blood donors (n = 20). Retrospectively selected clinical specimens tested by a commercial reference laboratory HBV sequencing assay (n = 54) or the Trugene HBV Genotyping kit (n = 7) and the Abbott HBV RUO Sequencing assay showed minor differences in detection and reporting of NA resistance-associated mutations in 7 of 61 (11.5%) specimens but complete agreement of genotype results. The Abbott HBV RUO Sequencing assay provided a convenient and efficient assay workflow suitable for routine clinical laboratory use, with the flexibility to be modified for customized detection of NA resistance-associated mutations, HBV genotype determination, and detection of immune escape mutations from a single contiguous HBV sequence.     

Performance of HBsAg quantification assays for detection of Hepatitis B virus genotypes and diagnostic escape-variants in clinical samples

BackgroundThe impact of hepatitis B virus (HBV) genomic variability on the measurement of HBsAg level has been poorly evaluated.ObjectiveThis study was designed to compare the performance of all the available assays measuring HBsAg level in this setting.Study designA large selection of wild type HBV genotypes (n = 184) and HBsAg strains harboring mutations in the S gene (n = 81) from clinical samples was studied with three HBsAg quantification assays: Architect HBsAg (Abbott), LiaisonXL Murex HBsAg Quant (DiaSorin) and the Elecsys HBsAgII (Roche).ResultsThe overall percentage of positive results was 99.2% for Abbott, 98.9% for DiaSorin and 98.1% for Roche. Abbott and Roche assays provided an excellent concordance in HBsAg quantification (global mean bias of −0.006 logIU/mL). By contrast, DiaSorin underestimated HBsAg level with values 0.112 logIU/ml and 0.103 logIU/ml lower than Abbott and Roche, respectively. By contrast, DiaSorin slightly over quantified gtC (2.5% over the expected value) while Abbott provided values 6.2% lower than expected and 16.2% lower than what observed for the other genotypes. HBsAg quantitative assays were influenced by HBs protein substitutions irrespective to the genotype but no specific protein pattern that would particularly impair the quantification by one technique has been identified. However, Roche seemed to be particularly impacted by substitutions at 145 residue: 75% of under quantified samples carried a substituted 145 residue.ConclusionThis head-to-head comparison indicates a good correlation between all current systems used to quantify HBsAg but clearly shows an influence of both the genotype and the presence of “a” determinant variants in the absolute quantification of HBsAg. While these discrepancies may not translate into major clinical consequence, they may explain an absence of detection of weak concentration of HBsAg on some systems.     

Performance of the DiaSorin LIAISON anti-HBs II for the detection of hepatitis B surface antibodies: Comparison with the Abbott Architect anti-HBs assay

BackgroundDetection of hepatitis B virus (HBV) surface antibodies (anti-HBs) is required in order to evaluate the response to hepatitis B vaccination and to optimize post-exposure monitoring. The widespread use of vaccines has highlighted the need for accurate and consistent quantification, yielding comparable quantitative results.ObjectivesThis study assessed the adequacy of DiaSorin LIAISON^® anti-HBs II assay in detecting anti-HBs antibodies and determined the correlation with Abbott Architect anti-HBs quantification.Study designAnti-HBs levels were measured in parallel using Abbott and DiaSorin kits on WHO international standard dilutions and 350 routine samples pre-screened with Abbott Architect anti-HBs. Three different serological panels were tested: vaccinated subjects (n = 121); subjects with past HBV infection (n = 109); and subjects with no HBV infection marker (n = 69). Serial dilutions from nine high-titer anti-HBs samples were used to address linearity.ResultsAnti-HBs concentration measured on WHO standard dilutions indicated a good calibration of DiaSorin values against international units, while results for Abbott were below those expected. A strong impact of the dilution matrix used was observed when performing dilutions on high-titer samples. Despite difference in absolute quantification, the overall clinical agreement between the two assays was 96.9%, with strong correlation (r = 0.92) between concentrations and good linearity over the quantification range.ConclusionsThe DiaSorin LIAISON^® anti-HBs II assay shows excellent standardization to the WHO standard and good linearity. The assay is suitable for current clinical laboratory practice.     

Performance of a new rapid test for the detection of hepatitis B surface antigen in various patient populations

BackgroundRapid diagnostic tests (RDT) have been developed for the detection of hepatitis B surface antigen (HBsAg). They represent a promising alternative to enzyme immunoassays and a powerful tool for large-scale screening and diagnosis of HBV infection, especially in regions without easy access to serological and molecular testing.ObjectivesThe aims of the present study were to evaluate the characteristics and clinical performance of a new CE-marked HBsAg RDT, DRW-HBsAg v2.0 assay (Diagnostics for the Real World™, Ltd., USA), in various patient populations, including those chronically infected with HBV, patients with severe acute hepatitis of unknown origin and pregnant women with unknown HBV serological status at delivery.ResultsThe lower limit of detection of the assay, evaluated in 21 clinical samples, ranged from 0.30 ± 0.07 to 0.97 ± 0.26 international units/mL (using Abbott Architect as a reference), depending on the HBV genotype. The assay tested positive in 100% of patients with chronic hepatitis B, 96.3% of HBsAg-positive acute hepatitis patients, and 95.2% of HBsAg-positive pregnant women. Its specificity was 98.8% in HBsAg-negative patients, 98.7% in HBsAg-negative patients with acute hepatitis of unknown origin and 97.8% in HBsAg-negative pregnant women. Amino acid substitutions in the HBsAg major hydrophilic region did not affect HBsAg detection by DRW-HBsAg v2.0.ConclusionsThe new DRW-HBsAg v2.0 assay is a simple, rapid, easy-to-run and highly sensitive assay that can be used in both high- and low-risk populations for the diagnosis of HBsAg carriage. It appears to be a promising new tool for large-scale screening and diagnosis of HBV infection.     

Evaluation of a new random-access HBV DNA molecular assay: The VERIS HBV assay

BackgroundDetection and quantification of HBV DNA are essential to diagnose chronic HBV infection, monitor the virological response to treatment and the possible selection of resistant viruses in order to tailor therapy. The VERIS/MDx System HBV Assay is a random-access system that quantifies HBV DNA in clinical samples using unique single sample and reagent access during the workflow process without the need to reload other tests and delivers results within 1.2 h following sampling.Objective and study designThe goal of this study was to evaluate the analytical performance of the VERIS HBV assay for HBV DNA detection and quantification in clinical samples from a series of patients chronically infected with different HBV genotypes.ResultsThe specificity of the VERIS HBV assay was estimated to be over 99.5%. The limit of detection (LOD) was estimated to be 4.1 IU/mL (95%CI: 3.20–5.90 IU/mL). Using an HBV linearity panel and controls (Seracare LifeScience), intra-assay and inter-assay coefficients of variation ranged from 0.12% to 3.64% and from 1.05% to 7.35%, respectively. The influence of the HBV genotype was evaluated from 120 clinical specimens containing HBV genotypes A to G tested in parallel with the VERIS HBV assay and the COBAS AmpliPrep/COBAS TaqMan HBV v2.0 assay. A linear relationship between the HBV DNA levels measured with both assays was found. A modest bias of HBV DNA levels was observed in the VERIS assay as compared to CAP/CTM HBV v2.0 in most of the samples tested (mean VERIS minus CAP/CTM difference: −0.395 log IU/mL). Overall, the VERIS HBV assay is well suited to monitoring clinical HBV DNA levels in infected patients according to current clinical practice guidelines.     

Correlation between the Elecsys HBsAg II assay and the Architect assay for the quantification of hepatitis B surface antigen (HBsAg) in the serum

BackgroundHepatitis B surface antigen (HBsAg) clearance during chronic hepatitis B (CHB) infection is associated with improved long-term clinical outcome, so is considered an important therapeutic goal in CHB. Studies have shown that serum HBsAg quantification during, and at end of, treatment may predict long-term HBsAg loss.ObjectivesPerformance comparison of the qualitative Elecsys HBsAg II assay using a quantitative research protocol and an established quantitative HBsAg assay.Study designA dilution algorithm was developed for the Elecsys HBsAg II assay to allow quantification of HBsAg levels; this was used to measure HBsAg levels in a range of samples including sera from patients infected with different HBV genotypes, HBV mutants, and longitudinal samples from patients undergoing antiviral treatment. Results were compared with those from the quantitative Architect HBsAg assay.ResultsThere was significant overall correlation between Elecsys and Architect assays (correlation coefficient [r] = 0.97; p < 0.001). HBsAg levels measured with both assays correlated well in all phases of infection (r = 0.80–0.96), across all genotypes tested (HBV genotype A, r = 0.89; HBV genotype D, r = 0.97), and in samples with lamivudine-resistant mutations (r = 0.94). Bland–Altman analysis showed only minor discordance between assays in different phases of chronic HBV-infection (3.8–5.1%). This strong correlation was also present for sera with lower HBsAg concentrations. On-treatment HBsAg levels were similar when measured with either assay.ConclusionsUsing a simple dilution algorithm, the quantitative Elecsys HBsAg II assay reliably determined serum HBsAg levels in a wide range of samples, and showed very high correlation with the Architect HBsAg assay.     

The variability of hepatitis B envelope is associated with HBs antigen persistence in either chronic or acute HBV genotype A infection

BackgroundMore than 240 million people are chronically infected by hepatitis B virus (HBV) worldwide. Envelope proteins play a crucial role in viral cellular entry and immune recognition. The loss of HBs antigen (HBsAg) correlated with a good clinical prognosis is rarely achieved with or without treatment (3–16%).ObjectivesHBV envelope variability was investigated according to HBsAg persistence.Study designThe cohort consisted of 15 HBV genotype A-infected patients divided into “resolvers”, with HBsAg clearance, and “non-resolvers”, with HBsAg persistence and in subgroups: acute (n = 5, AHBV) or chronic infection (n = 4, CHBV) and HBV/HIV coinfection (n = 6, CHBV/HIV). HBV S and preS sequences were studied by direct and ultra-deep sequencing. Amino acid sequences were analyzed with bioinformatics for predicted antigenicity.ResultsIn S gene, the complexity was lower in AHBV than in chronic-infected patients (p = 0.046). Major mutations, detected using direct sequencing, were more frequent in AHBV developing chronicity (p = 0.01) than in AHBV resolvers. In the Major Hydrophilic Region, more frequent mutations were observed in non-resolvers versus resolvers (p = 0.047) and non-resolvers tended to have more haplotypes with a reduced predicted antigenicity (p = 0.07). Most of the mutations in preS/S region were found rather in epitopic than in non-epitopic areas (p = 0.025). Interestingly, the mutation sY161F found in 3/8 non-resolvers was associated with a decrease in predicted antigenicity (28%; AnTheProt).ConclusionsHBsAg persistence was correlated with mutations and deletions in areas playing a key role in immune recognition. These data suggest that variability in HBV envelope could favor immune escape in various clinical settings of HBV genotype A-infected patients.     

The wide genetic landscape of clinical frontotemporal dementia: systematic combined sequencing of 121 consecutive subjects

PurposeTo define the genetic spectrum and relative gene frequencies underlying clinical frontotemporal dementia (FTD).MethodsWe investigated the frequencies and mutations in neurodegenerative disease genes in 121 consecutive FTD subjects using an unbiased, combined sequencing approach, complemented by cerebrospinal fluid Aβ_1-42 and serum progranulin measurements. Subjects were screened for C9orf72 repeat expansions, GRN and MAPT mutations, and, if negative, mutations in other neurodegenerative disease genes, by whole-exome sequencing (WES) (n = 108), including WES-based copy-number variant (CNV) analysis.ResultsPathogenic and likely pathogenic mutations were identified in 19% of the subjects, including mutations in C9orf72 (n = 8), GRN (n = 7, one 11-exon macro-deletion) and, more rarely, CHCHD10, TARDBP, SQSTM1 and UBQLN2 (each n = 1), but not in MAPT or TBK1. WES also unraveled pathogenic mutations in genes not commonly linked to FTD, including mutations in Alzheimer (PSEN1, PSEN2), lysosomal (CTSF, 7-exon macro-deletion) and cholesterol homeostasis pathways (CYP27A1).ConclusionOur unbiased approach reveals a wide genetic spectrum underlying clinical FTD, including 11% of seemingly sporadic FTD. It unravels several mutations and CNVs in genes and pathways hitherto not linked to FTD. This suggests that clinical FTD might be the converging downstream result of a delicate susceptibility of frontotemporal brain networks to insults in various pathways.     

Clinical course and core variability in HBV infected patients without detectable anti-HBc antibodies

BackgroundThe presence of anti-HBc antibodies indicates direct encounter of the immune system with hepatitis B virus (HBV).ObjectivesAim of our study was to seek for anti-HBc negative but HBV replicating patients and analyze their clinical course and preconditions.Study designFrom 1568 HBV-DNA positive patients, 29 patients (1.85%) tested negative for anti-HBc. The absence of anti-HBc could be confirmed in 19 patients using an alternative assay. In 16 of 19 cases, a partial or full HBV genome analysis was performed with NGS sequencing to evaluate if specific mutations were associated with anti-HBc absence. As a control group samples from 32 matched HBV infected patients with detectable anti-HBc were sequenced.ResultsPatients with detectable HBV-DNA and sequenced HBV core region in the confirmed absence of anti-HBc were diagnosed with acute HBV infection (n = 3), HBV reactivation (n = 9) and chronic hepatitis B (n = 4). Most patients (12/16) were immunosuppressed: 3/16 patients had an HIV coinfection, 7/16 patients suffered from a malignant disease and 4/16 patients underwent solid organ transplantation (from which 2/4 had a malignant disease). Compared to the control cohort, HBV variants from anti-HBc negative patients showed less variability in the core region.ConclusionsIn the absence of anti-HBc, HBV-DNA was most often found in immunocompromised hosts. Distinct mutations or deletions in the core region did not explain anti-HBc negativity. It would be advisable not to rely only on a single result of anti-HBc negativity to exclude HBV infection in immunocompromised hosts, but to measure anti-HBc repeatedly or with different methods     

Analytical and clinical performance characteristics of the Simplexa BK virus quantitative PCR assay for the diagnosis of polyomavirus-associated nephropathy in renal transplant recipients using plasma and urine specimens

BackgroundPolyomavirus-associated nephropathy is a significant cause of kidney rejection in renal transplant recipients. Quantification of BK viral load in plasma and urine can predict the development of polyomavirus-associated nephropathy, though each assay requires careful evaluation of analytical and clinical performance characteristics for optimal use.ObjectivesThis study evaluated the analytical and clinical performance characteristics of the Simplexa BK virus quantitative PCR assay.Study designAnalytical validation was performed using commercial standards, BK virus stock culture, and patient specimens. Clinical performance was evaluated using biopsy-proven BK nephropathy as the gold standard.ResultsThe Simplexa BK virus quantitative PCR assay was linear over a range of 2.7–10.4 log₁₀ copies/mL. Limit of detection was 2.7–2.8 log₁₀ copies/mL in plasma and urine samples. Sensitivities were 100% and 100% and specificities were 84% and 86% for plasma and urine samples, respectively, when compared to a reference BK assay. Clinical cutoff values of 4.0 log₁₀ copies/mL (plasma) and 7.5 log₁₀ copies/mL (urine) yielded 100% sensitivity and specificities of 87.5% and 85%, respectively, for biopsy-proven polyomavirus nephropathy.ConclusionsThe Simplexa BK virus quantitative PCR assay has high sensitivity and acceptable analytical characteristics for clinical use. The clinical cutoff values presented here provide a rational approach to the monitoring and treatment of renal transplant recipients for polyomavirus-associated nephropathy.     

Clinical performance of the novel DiaSorin LIAISON® XL murex: HBsAg Quant,HCV-Ab,HIV-Ab/Ag assays

BackgroundThe fully automated and closed LIAISON^®XL platform was developed for reliable detection of infection markers like hepatitis B virus (HBV) surface antigen (HBsAg), hepatitis C virus (HCV) antibodies (Ab) or human immunodeficiency virus (HIV)-Ag/Ab. To date, less is known about the diagnostic performance of this system in direct comparison to the common Abbott ARCHITECT^® platform.ObjectivesWe compared the diagnostic performance and usability of the DiaSorin LIAISON^®XL with the commonly used Abbott ARCHITECT^® system.Study designThe qualitative performance of the above mentioned assays was compared in about 500 sera. Quantitative tests were performed for HBsAg-positive samples from patients under therapy (n = 289) and in vitro expressed mutants (n = 37). For HCV-Ab, a total number of 155 selected samples from patients chronically infected with different HCV genotypes were tested.ResultsThe concordance between both systems was 99.4% for HBsAg, 98.81% for HCV-Ab, and 99.6% for HIV-Ab/Ag. The quantitative LIAISON^®XL murex HBsAg assay detected all mutants in comparable amounts to the HBsAg wild type and yielded highly reliable HBsAg kinetics in patients treated with antiviral drugs. Dilution experiments using the 2nd International Standard for HBsAg (WHO) showed a high accuracy of this test. HCV-Ab from patients infected with genotypes 1–3 were equally detected in both systems. Interestingly, S/CO levels of HCV-Ab from patients infected with genotype 3 seem to be relatively low using both systems.ConclusionsThe LIAISON^®XL platform proved to be an excellent system for diagnostics of HBV, HCV, and HIV with equal performance compared to the ARCHITECT^® system.     

Compartmental HBV evolution and replication in liver and extrahepatic sites after nucleos/tide analogue therapy in chronic hepatitis B carriers

BackgroundHepatitis B virus (HBV) variants are associated with nucleos/tide analogue (NA) response and liver disease but it is unknown whether NA influences extrahepatic HBV persistence.ObjectivesTo investigate HBV replication and genetic evolution in hepatic and extrahepatic sites of chronic hepatitis B (CHB) before and after NA therapy.Study DesignA total of 13 paired plasma, peripheral blood mononuclear cells (PBMC), were collected from chronic HBV carriers at baseline and after a median 53 weeks NA therapy as well as liver biopsy (N = 7 baseline, N = 5 follow-up). HBV covalently closed circular DNA (cccDNA) and messenger (m) RNA in liver and PBMC were analyzed. HBV polymerase (P)/surface (S), basal core promoter (BCP)/pre-core (PC)/C gene clonal sequencing was done in plasma, peripheral blood mononuclear cells (PBMC), and liver.ResultsCompare to baseline, at ∼53 weeks follow-up, there was no significant change in HBV cccDNA levels in liver (0.2–0.08 copies/hepatocyte, p > 0.05) or in PBMC 0.003–0.02 copies/PBMC, p > 0.05), and HBV mRNA remained detectable in both sites. At baseline, BCP variants were higher in PBMC vs. liver and plasma. After therapy, drug resistant (DR) and immune escape (IE) variants increased in liver but IE and PC variants were more frequent in PBMC. HBV P/S diversity was significantly higher in PBMC compared to plasma.ConclusionContinuous HBV replication occurs in liver and PBMC and shows compartmentalized evolution under selective pressure of potent NA therapy.     

Performance of HBsAg point-of-care tests for detection of diagnostic escape-variants in clinical samples

BackgroundHepatitis B viruses (HBV) harboring mutations in the a-determinant of the Hepatitis B surface antigen (HBsAg) are associated with reduced reactivity of HBsAg assays.ObjectivesTo evaluate the sensitivity and specificity of three HBsAg point-of-care tests for the detection of HBsAg of viruses harboring HBsAg mutations.Study designA selection of 50 clinical plasma samples containing HBV with HBsAg mutations was used to evaluate the performance of three HBsAg point-of-care tests (Vikia^®, bioMérieux, Marcy-L’Étoile, France. Alere Determine HBsAg™, Iverness Biomedical Innovations, Köln, Germany. Quick Profile™, LumiQuick Diagnostics, California, USA) and compared to the ARCHITECT HBsAg Qualitative^® assay (Abbott Laboratories, Sligo, Ireland).ResultsThe sensitivity of the point-of-care tests ranged from 98% to 100%. The only false-negative result occurred using the Quick Profile™ assay with a virus harboring a D144A mutation.ConclusionsThe evaluated point-of-care tests revealed an excellent sensitivity in detecting HBV samples harboring HBsAg mutations.     

Diagnostic performance of serological assays for anti-HBs testing: Results from a quality assessment program

BackgroundPost-vaccination testing after hepatitis B vaccination is indispensable to evaluate long-term immunological protection. Using a threshold level of antibodies against hepatitis B surface antigen (anti-HBs) to define serological protection, implies reproducible and valid measurements of different diagnostic assays.ObjectivesIn this study we assess the performance of currently used anti-HBs assays.Study designIn 2013, 45 laboratories participated in an external quality assessment program using pooled anti-HBs serum samples around the cutoff values 10 IU/l and 100 IU/l. Laboratories used either Axsym (Abbott Laboratories), Architect (Abbott Laboratories), Access (Beckman-Coulter), ADVIA Centaur anti-HBs2 (Siemens Healthcare Diagnostics), Elecsys, Modular or Cobas (Roche Diagnostics) or Vidas Total Quick (Biomerieux) for anti-HBs titre quantification. We analysed covariance using mixed-model repeated measures. To assess sensitivity/specificity and agreement, a true positive or true negative result was defined as an anti-HBs titre respectively above or below the cutoff value by ≥4 of 6 assays.ResultsDifferent anti-HBs assays were associated with statistically significant (P < 0.05) differences in anti-HBs titres in all dilutions. Sensitivity and specificity ranged respectively from 64%-100% and 95%-100%. Agreement between assays around an anti-HBs titre cutoff value of 10 IU/l ranged from 93%-100% and was 44% for a cutoff value of 100 IU/l.ConclusionsAround a cutoff value of 10 IU/l use of the Access assay may result in false-negative results. Concerning the cutoff value of 100 IU/l, a sample being classified below or above this cutoff relied heavily on the specific assay used, with both the Architect and the Access resulting in false-negative results.     

Detection and differentiation of HIV-2 using the point-of-care Alere q HIV-1/2 Detect nucleic acid test

BackgroundThe Alere q HIV-1/2 Detect test (Alere Detect) is a rapid point-of-care (POC) nucleic acid test (NAT) that can detect and differentiate HIV-1 and HIV-2 in 25-μL whole blood or plasma samples. The Alere Detect test has been validated for early infant diagnosis of HIV-1 infection, and it is the only POC NAT device currently known to detect HIV-2, which is endemic in West Africa.ObjectivesTo evaluate the sensitivity detecting HIV-2 RNA and the differential performance of the Alere Detect.Study designPlasma samples from non-HIV (n = 4), HIV-1 (n = 22), HIV-2 (n = 111; 29 Group A, 2 Group B) and HIV-1/HIV-2 dually-seropositive (n = 8) participants in Senegal and the United States and HIV-2 reference strains (3 Group A, 1 Group B) were tested by Alere Detect, Abbott RealTime HIV-1 and the University of Washington HIV-2 RNA quantitative (UW HIV-2) assays.ResultsThe Alere Detect correctly differentiated between HIV-1 and HIV-2 in all 80 (100%) patient samples with detectable HIV RNA (n = 20 HIV-1, 60 HIV-2). The overall HIV-2 detection concordance between Alere Detect and the UW HIV-2 assay was 68% (54/80); the concordance improved to 100% (30/30) for samples with HIV-2 RNA >300copies/mL. Neither assay detected HIV-2 RNA in 31 of 111 HIV-2 seropositive samples.ConclusionsThe Alere Detect test is a novel device detecting HIV RNA in clinical samples, and differentiating HIV-1 and HIV-2 with a high level of specificity. It has the potential for use as a rapid HIV-2 NAT-based diagnosis tool in resource-limited settings and to confirm HIV-2 infection for the CDC 4th generation HIV-1/2 diagnostic algorithm.     

Emergence of H274Y oseltamivir-resistant A(H1N1) influenza viruses in Japan during the 2008–2009 season

BackgroundA substantial increase in oseltamivir-resistant A(H1N1) influenza viruses was reported in Europe in late 2007.ObjectivesTo monitor the antiviral susceptibility profile of human A(H1N1) influenza viruses in Japan during the 2007–2008 and 2008–2009 seasons.Study designViruses were obtained from respiratory samples of patients with influenza collected in Japan between December 2007 and April 2008 (n = 1046) and between December 2008 and April 2009 (n = 1789). Oseltamivir resistance was determined by an H274Y-specific real-time PCR cycling probe assay and a neuraminidase inhibition assay. Amantadine resistance was assessed by sequencing the M2 gene. Sequencing of the hemagglutinin and NA genes was performed to infer phylogenetic relationships between different strains.ResultsThree of 687 (0.4%) A(H1N1) viruses from the 2007–2008 season and 745 of 745 (100%) viruses from the 2008–2009 season carried the NA–H274Y substitution and demonstrated a >300-fold reduction in oseltamivir susceptibility. All oseltamivir-resistant viruses from the 2008–2009 season possessed an A193T substitution in the receptor-binding domain of the hemagglutinin. Amantadine resistance was detected in 431 of 687 (62.7%) and 0 of 745 (0.0%) of the A(H1N1) viruses from the 2007–2008 and 2008–2009 seasons, respectively.ConclusionsA dramatic surge in oseltamivir-resistant A(H1N1) viruses possessing the NA–H274Y substitution was detected in Japan during the 2008–2009 season. The emergence of oseltamivir-resistant viruses was facilitated by mutations in the viral genome. Intensified surveillance, including phenotypic assays and sequencing of the hemagglutinin, neuraminidase, and M2 gene would allow monitoring of the spread and evolution of drug-resistant influenza virus variants.     

Comparison of three different hybridization assays in the quantitative measurement of serum hepatitis B virus DNA

The measurement of hepatitis B virus (HBV) DNA, is important for monitoring and evaluating the efficacy of anti-viral agents in the treatment of patients with chronic hepatitis B. Three different hybridization assays for quantitative measurement of HBV DNA: direct membrane (dot-blot) hybridization, liquid hybridization (Abbott HBV DNA assay) and branched DNA signal amplification assay (Quantiplex^TM, Chiron), were applied to 114 serial serum samples obtained from 13 patients with chronic active hepatitis B who had received ribavirin 600 mg daily for four weeks. Among the three assays, the correlation was found to be highest between Quantiplex and Abbott HBV DNA assay (r = 0.71, p < 0.01). moderate between Quantiplex and dot-blot hybridization (r = 0.58, p < 0.01) and lowest between dot-blot hybridization and Abbott HBV DNA assay (r = 0.27, p < 0.01). Quantiplex detected 107 (94%) of 114 specimens and was the most sensitive assay. All specimens positive by dot-blot hybridization and Abbott HBV DNA assays were detected positive by Quantiplex. The Dot-blot hybridization assay detected all 89 (100%) specimens with a high HBV DNA level (≥ 10 million genome equivalent (Meq)/ml by Quantiplex), but detected only 7 (50%) of 14 specimens with a low HBV DNA level ( < 10 Meq/ml). The Abbott HBV DNA assay detected 85 (95%) of 89 specimens with a high HBV DNA level, but detected only 3 (17%) of 18 specimens with a low HBV DNA level. Among 7 negative specimens in the Quantiplex assay, 2 were detected positive by polymerase chain reaction. In conclusion, Quantiplex assay was more sensitive than Abbott HBV DNA assay and dot-blot hybridization assay for quantitative measurement of serum HBV DNA and can be used in the evaluation of the therapeutic drug effect on chronic hepatitis B patients.     

High prevalence of primary Enfuvirtide (ENF) resistance-associated mutations in HIV-1-infected patients in Hong Kong

BackgroundEnfuvirtide (ENF) is a viral fusion inhibitor used in patients failing highly active antiretroviral therapy (HAART). Mutations associated with ENF resistance have been identified within amino acid positions 36–45 of gp41. As ENF will be introduced to Hong Kong, an understanding of the prevalence of naturally occurred ENF resistance mutations is important before implementation of ENF treatment.ObjectivesTo investigate the prevalence of ENF resistance-associated mutations in the HR1 and HR2 of HIV-1 strains obtained from ENF-naïve patients.Study designHIV-1 strains isolated from 185 patients (156 antiretroviral treatment [ART]-naïve and 29 HAART-experienced) were screened for ENF resistance-associated mutations using RT-PCR and DNA sequencing.ResultsPrimary mutations were detected in 19.4% of HARRT-experienced patients and 20.5% of ART-naïve patients. G36D was encountered most frequently and more prevalent in non-B subtypes. N42S, L54 M and V69I were the major polymorphisms detected. N42S and L54M were predominant in CRF01_AE and subtype B, respectively. V69I was found in all samples harboring G36D. In three longitudinal samples from an ENF-treated patient, G36D was detected after ENF treatment for 6 months and the mutation persisted after termination of ENF for 6 months.ConclusionsThe high prevalence of ENF resistance-associated mutations in HARRT-experienced and ART-naïve patients identified in this study highlights the importance of mutation screening before ENF therapy in Hong Kong. Our findings from the ENF-treated patient showed that G36D mutation persisted as long as 6 months after ENF withdrawal. Phenotypic assays will be necessary to confirm the influence of this mutation to ENF susceptibility.     

Comparative performance of the new Aptima HIV-1 Quant Dx assay with three commercial PCR-based HIV-1 RNA quantitation assays

BackgroundQuantitative measurement of HIV-1 RNA levels in plasma (‘viral load’) plays a central role in clinical management. The choice of assay platform can influence results and treatment decisions.ObjectiveTo compare the analytical performance of the new TMA-based Hologic Aptima^® HIV-1 Quant Dx assay with that of three PCR-based assays: Abbott RealTime HIV-1, Qiagen Artus^® HI Virus-1 QS-RGQ, and Roche CAP/CTM HIV-1 Test v2.Study designAssay performance was evaluated using Acrometrix HIV-1 RNA Standard panels; the 3rd WHO HIV-1 RNA International Standard (12–500 copies/ml; 6 dilutions; 9 replicates); and plasma samples from 191 HIV-positive patients.ResultsAptima showed high (>0.99) precision, accuracy and concordance with the Acrometrix Standards across a wide dynamic range (2.0–6.7 log₁₀ copies/ml). Variance caused up to 2.1 (Aptima), 1.7 (RealTime), 7.5 (Artus), and 1.9 (CAP/CTM) fold changes in the International Standard quantifications at 50–500 copies/ml. HIV-1 RNA detection rates in plasma samples were 141/191 (74%), 119/191 (62%), 108/191 (57%), and 145/191 (76%) for Aptima, RealTime, Artus and CAP/CTM, respectively. For categorising samples either side of 50 copies/ml, Aptima had excellent agreement with RealTime (kappa 0.92; 95% CI 0.87–0.98); lowest agreement was with Artus (kappa 0.79; 95%CI 0.70–0.88). Aptima quantifications were mean 0.12 and 0.06 log₁₀ copies/ml higher compared with RealTime and CAP/CTM, respectively, and 0.05 log₁₀ copies/ml lower compared with Artus. Limits of agreement were narrowest when comparing Aptima to RealTime.ConclusionsThe new Aptima HIV assay is sensitive, precise, and accurate. HIV assays exhibit discordance at low HIV-1 RNA copy numbers.