Antiviral Drug Resistance Mutations Research Articles

Potential antiviral drug resistance mutations in patients with treatment-naive chronic hepatitis B

Potential antiviral drug resistance mutations in patients with treatment-naive chronic hepatitis B

Phylogenetic analysis and docking study of neuraminidase gene of influenza A/H1N1 viruses circulating in Iran from 2010 to 2019

Influenza A viruses (H1N1) have been consistently one of the most evolving viruses that escape from vaccine-induced immunity. Although there has been a rapid rise in human influenza virus knowledge since the 2009 pandemic, the molecular information about Iranian strains is still inadequate. The aim of this study was to analyze the neuraminidase (NA) segment of the Iranian isolates in terms of phylogenetic, antiviral resistance, and vaccine efficiency. Ninety-three NA sequences collected among 1758 nasopharyngeal swab samples during the 2015–2016 influenza season were sequenced and submitted to NCBI. Moreover, all the submitted Iranian influenza H1N1 NA sequences since 2010 till 2019 were included in the study.Software including MEGA-X, MODELLER, UCSF ChimeraX, Auto-Dock 4.2, and other online tools were used to analyze the phylogenetic relationship, vaccine efficiency, and binding affinity to sialic acid of the selected NA proteins. Moreover, the information about antiviral drug resistance mutations of NA were gathered and compared to the Iranian NA segments to check the presence of antiviral drug-resistant strains.The phylogenetic study showed that most Iranian NA sequences (between 2015 and 2016) were located in a single clade and following years were located in its subclade by 3 major mutations (G77R/K, V81A, and J188T). Resistant mutations in drug targets of NA including I117M, D151E, I223V, and S247N were ascertained in 10 isolates during the 2015–2016 flu seasons.Investigation of vaccination effect revealed that Iranian isolates in 2017 and 2018 were best matched to A/Brisbane/02/2018 (H1N1), and in 2019 to A/Guangdong-Maonan/SWL1536/2019 (H1N1).Furthermore, we performed an in-silico analysis of NA enzymatic activity of all Iranian sequences by assessment of enzyme stability, ligand affinity, and active site availability. Overall, the enzyme activity of four Iranian strains (AUG84119, AUG84157, AUG84095, and AUG84100) was assumed as the maximum enzyme activity. This study highlighted the evolutionary trend of influenza A virus/H1N1 circulating in Iran, which provides a preliminary viewpoint for a better comprehension of new emerging strains’ virulence and thus, more appropriate monitoring of influenza virus A/H1N1 during each outbreak season.

Fast and Sensitive Real-Time PCR Detection of Major Antiviral-Drug Resistance Mutations in Chronic Hepatitis B Patients by Use of a Predesigned Panel of Locked-Nucleic-Acid TaqMan Probes.

We developed a novel real-time PCR assay that simultaneously evaluates 11 major nucleos(t)ide antiviral (NA) drug resistance mutations (mt) in chronic hepatitis B patients (CHB), including L180M, M204I/V, and V207M (lamivudine [LMV] resistance), N/H238A/T (adefovir [ADF] resistance), which are circulating in Vietnam; and T184G/L, S202I, and M250V (entecavir [ETV] resistance) and A194T (tenofovir resistance), which have been recently reported in several studies across the globe. We detected drug-resistant mt in hepatitis B virus (HBV) samples using our predesigned panel of allele-specific locked-nucleic acid (LNA) probes. Our assay had a high sensitivity of 5% in a low-HBV DNA population of ≥5 × 103 IU/ml and was validated in a cohort of 130 treatment-naive children and 98 NA-experienced adults with CHB. Single-point mt for LMV and ADF resistance were detected in 57.7% and 54.1% of the child and adult samples, respectively, with rtV207M (children, 42.3%; adults, 36.7%) and rtN238T/A (children, 15.4%; adults, 16.3%) being the most frequent mt in these populations. Multiple-point mt, including rtL180M-rtM204V- rtN238A and rtL180M-rtM204I, were identified in only two children, resulting in LMV-ADF resistance and reduced ETV susceptibility. In conclusion, this assay accurately identified the mt profile of children (98.4%) and adults (91.2%) with CHB, which is comparable to established methods. This fast and sensitive screening method can be used for the detection of major NA-resistant mt circulating in developing countries, as well as providing a model for the development of similar mt-detection assays, especially for use in nonhospitalized patients who need their results within half a day, before starting treatment.

Molecular Characterization of Hepatitis C Virus for Developed Antiviral Agents Resistance Mutations and New Insights into in-silico Prediction Studies.

BackgroundIdentification and characterization of developed antiviral drug resistance mutations are key to the success of antiviral therapies against hepatitis C virus (HCV), which remains a worldwide highly prevalent pathogenic disease. Although most studies focus on HCV genotypes 1, 2 or 3, the investigation of drug resistance in HCV genotype 4, predominant in North Africa, is especially significant in Egypt.MethodsWe performed mutational and genotypic analysis of the untranslated region (UTR) and nonstructural protein 5B (NS5B) drug resistance-associated regions of HCV for patients in the surrounding villages of Mansoura city, who were not responding to different antiviral treatments (sofosbuvir (SOF), ribavirin, and interferon). Furthermore, molecular modelling approaches (homology modelling and docking studies) were used to investigate the significance of the identified NS5B mutations for SOF and ribavirin binding in the HCV genotype 4a NS5B active site.ResultsGenotypic analysis confirmed all samples to have genotype 4 with sub-genotype 4a predominant. Partial sequencing of the UTR and NS5B resistance-associated regions identified D258E, T282S and A307G mutations in all isolates of NS5B. The UTR mutation site at position 243 was associated with interferon resistance, whereas the NS5B T282S mutation was considered as significant for SOF and ribavirin resistance. Docking studies in the HCV genotype 4a homology model predict SOF and ribavirin to accommodate a nucleotide-like binding mode, in which the T282 residue does interfere with the binding as it would in HCV genotypes 1 and 2. Mutation energy calculations predict T282S to moderately destabilize the binding of SOF and ribavirin by 0.57 and 0.47 kcal/mol, respectively.ConclusionThe performed study identified and characterized several antiviral drug resistance mutations of HCV genotype 4a and proposed a mechanism by which the T282S mutation may contribute to SOF and ribavirin resistance.

COLD-PCR Method for Early Detection of Antiviral Drug-Resistance Mutations in Treatment-Naive Children with Chronic Hepatitis B.

We investigated Nucleos(t)ide-analogue (NA)-resistance mutations (mt) in 142 treatment-naive children with Chronic Hepatitis B (CHB), using a sensitive co-amplification at lower denaturation temperature (COLD)-PCR with Sanger DNA sequencing. An NA resistance-associated mt in the hepatitis B virus (HBV) reverse transcriptase (RT) was found in 66.2% of the patients, with nonclassical mt contributing the most (64.8%). Significantly higher frequencies of Lamivudine (LMV) and Adefovir dipivoxil (ADF) resistance-associated mt were found in genotypes B and C, respectively (ORLMV/ADF: 1495.000; 95% CI: 89.800–24,889.032; p < 0.001). Single-point mt associated to LMV and ADF resistance were detected in 59.9% of the tested children with rtV207M (38.0%) and rtN238T (9.9%) being the most frequent. Multiple-point mt were found only in 8 cases (5.6%): 6 children carried double mt (rtV207M + rtL229V; rtV207M + rtI233V; rtV207I + rtV207M × 2 cases; rtV207M + rtS213T; rtN238A + rtS256G) relating to LMV or/and ADF resistance and 3 children carried triple mt (rtL180M + rtM204I + rtN238T; rtV207M + rtS213T + rtS256G) or quadruple mt (rtL180M + rtM204V + rtV207I/M) for LMV-ADF resistance and Entecavir-reduced susceptibility. Our data indicate that significantly higher frequencies of LMV and ADF-associated mutations were found in treatment-naïve children infected with HBV genotypes B and C, respectively. The developed COLD-PCR method and obtained data may contribute to the development of suitable treatments for children with CHB.

Performance of NS3, NS5a AND NS5b Hepatitis C Virus (HCV) Antiviral Resistance Sequencing Assays

BackgroundHCV genotype 1a (HCV g1a) followed by HCV g1b, –g2, and –g3 are the most common etiologic agents in the ~3 million current HCV-infections in the US. To achieve effective therapy, antiviral drug resistance testing is often essential but not fully available. Knowledge of both the genotype and the presence of HCV mutations in the genes of the major drug targets (NS3, NS5A and NS5B) can assist in optimal treatment selection.MethodsThe HCV genotype of 1000 HCV positive clinical plasmas and sera was determined (HCVg Direct, GenMark). Ten independent Sanger sequencing assays detecting antiviral drug resistance mutations in the genes encoding NS3, NS5a, and NS5b were developed. Six of these assays address mutations in all three genes in the two most common genotypes (HCV g1a and g1b). In addition, four more assays address mutations in NS5a and NS5b of HCV g2 and g3. These mutations are resistance determinants against 11 anti-HCV drugs as shown in Figure 1. A streamlined workflow employs conventional reverse transcriptase PCR, gel electrophoresis, spectrophotometry, bi-directional Sanger sequencing and reporting. The assays were designed to cover hot spot regions and capturing all known resistance mutations in NS3, NS5a and NS5b.ResultsConsistent with previous US HCV incidence reports, g1a, g1b, g2, and g3 comprised 99% of 1000 sequentially tested HCV patient specimens (62%, 12%, 11%, and 14%, respectively). Testing of more than 20 clinical samples each for g1a, g1b, g2, and g3 resulted in successful detection of NS3, NS5a, and NS5b mutations that confer drug resistance. The design successfully permitted detection of relevant mutations known to date for all 11 drugs. The number of reportable mutations range from 20 ‒ 36, 9 ‒ 49, and 10 ‒ 29 for the NS3, NS5a, and NS5b inhibitors, respectively (Figure 1).ConclusionThese assays provide the most comprehensive commercially available antiviral drug resistance information to date for mutations in HCV NS3, NS5A, and NS5B. This testing will assist physicians in deciding on the most appropriate treatment options for their patients.Disclosures J. Nutt, Viracor Eurofins Laboratories: Employee, Salary; J. Grantham, Viracor Eurofins Laboratories: Employee, Salary; M. Smith, Viracor Eurofins Laboratories: Employee, Salary; E. Smith, Viracor Eurofins Laboratories: Employee, Salary; A. Wedin, Viracor Eurofins Laboratories: Employee, Salary; A. Tyler, Viracor Eurofins Laboratories: Employee, Salary; M. Miralles, Viracor Eurofins Laboratories: Employee, Salary; S. Kleiboeker, Viracor Eurofins Laboratories: Employee, Salary; M. Wissel, Viracor Eurofins Laboratories: Employee, Salary

New universal primers for genotyping and resistance detection of low HBV DNA levels.

HBV (hepatitis B virus) genotyping is important in determining the clinical manifestation of disease and treatment response, particularly, in patients with low viral loads. Also, sensitive detection of HBV antiviral drug resistance mutations is essential for monitoring therapy response.Asensitive direct sequencing method for genotyping and the drug resistance mutation detection of low levels of HBV DNA in patients’ plasma is developed by PCR amplification of the DNA with novel universal primers.The novel, common, and universal primers were identified by alignment of RT region of all the HBV DNA sequences in databases. These primers could efficiently amplify the RT region of HBV virus at low DNA levels by directly sequencing the resulting PCR products, and mapping with the reference sequence made it possible to clearly obtain the HBV subtypes and identify the resistance mutations in the samples with HBV DNA level as low as 20 IU/mL. We examined the reliability of the method in clinical samples, and found it could detect the HBV subtypes and drug resistance mutations in 80 clinical HBV samples with low HBV DNA levels ranging from 20 to 200 IU/mL.This method is a sensitive and reliable direct sequencing method for HBV genotyping and antiviral drug resistance mutation detection, and is helpful for efficiently monitoring the response to therapy in HBV patients.

Quality evaluation and clinical applicability of pyrosequencing assay kit for detecting hepatitis B virus resistance

To evaluate the quality and clinical applicability of pyrosequencing assay kit for detecting hepatitis B virus resistance (HBV DRT). Serial dilutions of the International Standard for HBV DNA were used to test the detection limit of the PCR for HBV DRT. Plasmids containing the either a wild-type (WT) copy or one of 10 mutant (MT) copies of the HBV RT gene were used to prepare a series of samples with various mutation ratios. To construct the linear relationship between the true mutation rate and the detected mutation rate, each sample was repeated at least 10 times. A total of 102 clinical samples were analyzed by Sanger sequencing and retested by the PCR for HBV DRT to determine the concordance of these two methods. The lower detection limit of the PCR for HBV DRT was 50 IU/ml. Except for the RT236 MT, the correlation between the true mutation rate and the detected mutation rate for the other nine resistance-related mutation sites were excellent, with R² more than 0.98 (P less than 0.001). Among the 102 clinical samples, four were not amplified successfully by PCR. The results were significantly different between the PCR for HBV DRT method and the Sanger sequencing method (x² = 71.2, P less than 0.001), and concordance was observed for 897/969 (92.6%) amino acid positions in 98 samples. Concordant results were achieved in 46/98 (46.9%) samples at all 10 mutation sites. For detection of a single mutation site, concordance rates ranged from 71.5% to 100% at the 10 mutation sites, respectively. Analysis of discordant samples showed that in 87.5% (63/72), Sanger sequencing detected WT and the PCR for HBV DRT detected WT/MT. In 5.6% (4/72) of samples, Sanger sequencing detected WT/MT and the PCR for HBV DRT detected WT. In the remaining 6.9% (5/72) of samples, Sanger sequencing detected WT but PCR for HBV DRT detected MT. The PCR for HBV DRT showed high sensitivity and accuracy in detecting antiviral drug-resistant mutations. The method is superior to Sanger sequencing for detecting minor mutations and can be used for early detection of a resistance mutation.

A description of the hepatitis B virus genomic background in a high-prevalence area in China.

BackgroundHepatitis B (HB) is an important disease worldwide. Almost 350 million people are positive for Hepatitis B virus surface antigen (HBsAg), and one-third of them live in China. According to a nation-wide serosurvey in China in 2006, the prevalence of HBsAg was higher in Northwest China than in other areas. However, the epidemic HBV strains in this area are poorly studied.ResultsIn this study, 242 complete hepatitis B virus (HBV) genome sequences were obtained from HBV asymptomatic carriers in major cities of Northwest China. The 242 HBV sequences clustered into genotypes B, C and D. Through comparison of the genotype consensus sequences, 158 genotype-dependent positions were observed in P, S and X ORFs. Clinically relevant mutation screening in this study revealed that no HBV antiviral drug resistance mutations were observed and the vaccination failure mutations were heavily underrepresented.ConclusionsThe role of genotype D strains in HBV prevalence should not be ignored in Northwest China. Due to low prevalence of vaccination failure mutations, it can be inferred that the genotype B, C and D strains in Northwest China may have less likelihood of vaccine escape.

Analysis of potential antiviral resistance mutation profiles within the HBV reverse transcriptase in untreated chronic hepatitis B patients using an ultra-deep pyrosequencing method

The potential antiviral resistance mutations within hepatitis B virus (HBV) reverse transcriptase (RT) region for nucleos(t)ide analogues (NA) are not well known. Especially, the effect of pre-existing antiviral drug resistance mutations in untreated patients in comparison to the resistance developed after treatment is not still clear. Sixteen naive chronic hepatitis B patients were studied. None of the patients had received NA treatment prior to the serum samples being collected. Forty-two potential NA resistance (NAr) mutation sites were screened by ultra-deep pyrosequencing (UDPS). After therapy, mutations conferring treatment resistance were detected by LiPA. Serum samples taken before treatment showed no classic primary or compensatory/secondary drug resistance mutations. However, NAr mutations found in 6 isolates (37.5%) involved 7 positions including rtL91I, rtT128I, rtQ215P, rtF221Y, rtN238D, rtC256S, and rtI266G. Substitutions at 3 NAr mutation sites (rtT128I, rtN238D, and rtC256S) were detected in 3 unresponsive patients developing drug resistance after NA treatment. One patient with rtI266G mutation also developed drug resistance after lamivudine (LAM) therapy. However, the relationship between rtI266G mutation and NA drug resistance was not previously reported. These results suggest that association of potential mutations besides the primary and secondary/compensatory resistance mutations should be investigated. Investigation of NAr mutations before treatment may be important for the success of the treatment.

Rapid Detection of Hepatitis B Virus Variants Associated with Lamivudine and Adefovir Resistance by Multiplex Ligation-Dependent Probe Amplification Combined with Real-Time PCR

Drug-resistant mutations of hepatitis B virus (HBV) are the major obstacles to successful therapy for chronic hepatitis B infection. Although there are many methods for detecting the antiviral drug-resistant mutations of HBV, their applications are restricted because of their shortcomings, such as low sensitivity, the time required, and the high cost. For this study, a multiplex ligation-dependent probe real-time PCR (MLP-RT-PCR) method was developed to simultaneously detect lamivudine (LAM)- and adefovir (ADV)-resistant HBV mutants (those with the mutations rtM204V/I, rtA181V/T, and rtN236T). The new method combined the high-throughput nature of multiplex ligation-dependent probe amplification (MLPA) with the rapid and sensitive detection of real-time PCR. In this report, MLP-RT-PCR was evaluated by detecting drug-resistant mutants in 116 patients with chronic hepatitis B infection. By MLP-RT-PCR analysis, LAM-resistant mutations were detected in 41 patients (35.3%), ADV-resistant mutations were detected in 17 patients (14.7%), and LAM- and-ADV-resistant mutations were detected in 5 patients (4.3%). Based on the results of MLP-RT-PCR, the mutations rtM204V, rtM204I, rtA181T, rtA181V, and rtN236T were 95.7% (111/116 patients), 98.3% (114/116 patients), 99.1% (115/116 patients), 98.3% (114/116 patients), and 99.1% (115/116 patients) concordant, respectively, with those of direct sequencing. The MLP-RT-PCR assay was more sensitive than direct sequencing for detecting mutations with low frequencies. Four samples containing the low-frequency (<10%) mutants were identified by MLP-RT-PCR and further confirmed by clonal sequencing. MLP-RT-PCR is a rapid and sensitive method that enables the detection of multidrug-resistant HBV mutations in clinical practice.

Severe acute hepatitis B in a treatment‐naïve patient with antiviral drug resistant mutations in the polymerase gene

This is a case of 62 years old Caucasian treatment-naïve patient who developed a severe acute hepatitis B infection soon after a trip to Thailand. The infection was due to genotype C HBV which was found to be resistant to lamivudine and telbivudine. The patient was treated with tenofovir resulting in complete suppression of viral replication and complete clinical and laboratory remission of acute hepatitis. Later the patient also developed seroconversion of HBeAg to anti-HBe and of HBsAg to anti-HBs. This case demonstrates that mutations of HBV polymerase associated with lamivudine, telbivudine, and adefovir resistance can be present also in untreated patients with severe acute hepatitis B. This suggests that in the clinical context, which represents a life threatening condition, a baseline resistance-testing should be an additional marker in the diagnostic evaluation process. Finally, this case report seems to support the use of tenofovir for the immediate treatment of severe acute hepatitis B.

Viral Hepatitis in Liver Transplantation

Liver transplantation is the only alternative for patients with end-stage liver disease. Viral hepatitis B and C are among the most common causes of cirrhosis and hepatocellular carcinoma and a frequent indication for liver transplantation. Hepatitis B virus immunoglobulin and nucleot(s)ide analogues have facilitated the management of patients with hepatitis B who have received liver transplants and resulted in excellent long-term outcomes. On the contrary, recurrence of hepatitis C is the main cause of graft loss in most transplant programs. Current therapeutic approaches are far from optimal, because sustained virologic responses are only achieved in one-third of treated patients, and adverse effects are common and severe. However, the rapid development of direct-acting antivirals against hepatitis C virus will change the management of this disease and in a few years prevent graft infection with this virus.

Applications of Next-Generation Sequencing Technologies to Diagnostic Virology

Novel DNA sequencing techniques, referred to as “next-generation” sequencing (NGS), provide high speed and throughput that can produce an enormous volume of sequences with many possible applications in research and diagnostic settings. In this article, we provide an overview of the many applications of NGS in diagnostic virology. NGS techniques have been used for high-throughput whole viral genome sequencing, such as sequencing of new influenza viruses, for detection of viral genome variability and evolution within the host, such as investigation of human immunodeficiency virus and human hepatitis C virus quasispecies, and monitoring of low-abundance antiviral drug-resistance mutations. NGS techniques have been applied to metagenomics-based strategies for the detection of unexpected disease-associated viruses and for the discovery of novel human viruses, including cancer-related viruses. Finally, the human virome in healthy and disease conditions has been described by NGS-based metagenomics.

Synthetic long oligonucleotides to generate artificial templates for use as positive controls in molecular assays: drug resistance mutations in influenza virus as an example

BackgroundPositive controls are an integral component of any sensitive molecular diagnostic tool, but this can be affected, if several mutations are being screened in a scenario of a pandemic or newly emerging disease where it can be difficult to acquire all the necessary positive controls from the host. This work describes the development of a synthetic oligo-cassette for positive controls for accurate and highly sensitive diagnosis of several mutations relevant to influenza virus drug resistance.ResultsUsing influenza antiviral drug resistance mutations as an example by employing the utility of synthetic paired long oligonucleotides containing complementary sequences at their 3' ends and utilizing the formation of oligonucleotide dimers and DNA polymerization, we generated ~170bp dsDNA containing several known specific neuraminidase inhibitor (NAI) resistance mutations. These templates were further cloned and successfully applied as positive controls in downstream assays.ConclusionThis approach significantly improved the development of diagnosis of resistance mutations in terms of time, accuracy, efficiency and sensitivity, which are paramount to monitoring the emergence and spread of antiviral drug resistant influenza strains. Thus, this may have a significantly broader application in molecular diagnostics along with its application in rapid molecular testing of all relevant mutations in an event of pandemic.

Evaluation of the performance of a DNA microarray method for detecting HBV mutations conferring antiviral resistance to lamivudine and adefovir

Objective To evaluate the performance of a DNA microarray method for detecting HBV antiviral drug-resistant mutations. Methods Two hundred and twenty four serum samples from patients with CHB were tested in parallel by DNA microarray and direct sequencing for the mutations within the HBV reverse transcriptase (rt) region, which included rtL180, rtA181, rtM204 and rtN236. Samples with discrepant results were retested by clonal sequencing. Results Complete concordance between DNA microarray and direct sequencing results was observed in 214 out of 224 samples (95. 5% ). The presence of mixed viral populations in the other 10 samples detected by DNA microarray but not by direct sequencing was confirmed by clonal analysis. The DNA microarray could detect minor viral populations which constituted 5.0%-15. 0% of the total viral load. Conclusion DNA microarray is highly consistent with direct sequencing in detecting HBV mutations conferring drug resistance and more sensitive in detecting mixed mutant and wild-type sequences than direct sequencing, which makes it a useful tool for early detection of drug resistance early. Key words: Hepatitis B,chronic; Hepatitis B virus; Oligonucleotide array sequence analysis; Drug resistance, viral; Sequence analysis, DNA

PCR with quenching probes enables the rapid detection and identification of ganciclovir-resistance-causing U69 gene mutations in human herpesvirus 6

A single-nucleotide polymorphism detection assay using PCR with quenching probes (QP-PCR) was developed for the rapid detection of antiviral drug-resistance mutations of human herpesvirus 6 (HHV-6). The mutations examined were in the HHV-6 U69 gene, and were single-base mutations in sequences known to be associated with ganciclovir (GCV) resistance in HCMV. We previously confirmed that they conferred GCV resistance to recombinant baculoviruses (Nakano et al., J. Virol. Methods 161:223–230, 2009). Six characterized mutations, including a previously reported one that encodes a GCV-sensitive kinase-activity mutant (Isegawa et al., J. Clin. Virol. 44:15–19, 2009), were used. The six mutations were separated into three groups based on their location in the U69 protein, and detected by the hybridization of three probes. We developed and validated a set of assays for these mutations using PCR followed by differential melting of a fluorescently labeled oligo probe, on a Roche Light Cycler platform. Nucleobase quenching was used to detect the hybridized probe. The optimized assay could distinguish the different mutants, and easily detected mutants representing 30% of the DNA in a mixed sample. This QP-PCR assay permitted the rapid (1.5 h), objective, and reproducible detection of drug-resistant mutations of HHV-6.

Validation of the INNO-LiPA HBV DR Assay (Version 2) in Monitoring Hepatitis B Virus-Infected Patients Receiving Nucleoside Analog Treatment

Hepatitis B virus (HBV) antiviral drug resistance mutations prevent successful outcome of treatment and lead to worsening of liver disease. Detection of its emergence permits opportune treatment with alternative drugs. Unfortunately, the use of newly approved antivirals, including adefovir dipivoxil, emtricitabine, and telbivudine, is also associated with the development of drug resistance, albeit to a lesser extent than the use of lamivudine. The objectives of this work were to assess the performance characteristics (sensitivity and accuracy) of an updated drug resistance test, the INNO-LiPA HBV DR v2, which includes detection of mutations associated with lamivudine, adefovir, emtricitabine, and telbivudine resistance, and to compare the results with consensus sequencing of serum samples from patients treated with HBV antivirals. Diagnostic sensitivity, defined as detection of a positive amplification line on the line probe assay (LiPA) strip, was 94.8% (95% confidence interval [CI], 89.7 to 97.9) after initial testing, increasing to 96.3% (95% CI, 91.6 to 98.8) after repeat test 1 and to 100% (95% CI, 97.3 to 100.0) after repeat test 2. In diagnostic accuracy determinations, full concordance was observed between sequencing and LiPA for 77.0% of the codons tested (620/805 codons [95% CI, 74.0 to 79.9]), whereas LiPA and sequencing were partially concordant 22% of the time (177/805 codons). In 167 out of 177 cases, LiPA detected a wild-type/mutant mixture whereas sequencing detected only one of the two results. Performance testing of the new LiPA test, the INNO-LiPA HBV DR v2, showed convincing diagnostic sensitivity and accuracy. The ability of the test to detect mixed infections and minority viral populations associated with resistance to the current generation of antivirals, including adefovir, emtricitabine, and telbivudine, makes it a useful tool for HBV therapy monitoring.

Designing a low-cost drug resistance database for viral hepatitis

Plans are outlined for a new database for hepatitis antiviral drug resistance mutations that will be closely linked to existing sequence databases. The new database will contain an infrastructure to store all available information on drug resistance mutations for the virus and a mutation can be searched by itself or in conjunction with the sequence information. Self-maintaining and self-updating sequence databases already exist for several viruses. The resistance mutation database would be annotated by authorized users, recruited from among clinicians and researchers familiar with each virus. The new database would provide a central location to find known drug resistance mutations for HBV and HCV; the ability to search the sequence database for each (combination of) mutations, retrieve alignments of relevant sections, and analyse mutation context and other background information; and rapid inclusion of newly found mutations without the need to wait for a publication.

PV-2 Performance evaluation of the Access® HIV-1/2 new assay performed on the UniCel® DxI 800 in a virology laboratory

Microarrays of DNA probes have at least three roles in clinical virology. These are: firstly, in diagnosis, to recognise the causative agent of an illness; secondly, for molecular typing for (i) patient management, (ii) epidemiological reasons (e.g. investigating routes of transmission), (iii) purposes related to vaccine use; and thirdly, in research, to investigate the interactions between the virus and the host cell. Microarrays intended for syndromic diagnostic purposes require genome specific probes to capture the unknown target viral sequences and thereby reveal the presence of that virus in a test sample. Microarrays intended for typing and patient management, e.g. monitoring antiviral drug resistant mutations require a set of probes representing the important sequence variants of one or more viral genes. Microarrays intended for research into virus–host interactions require probes representative of each individual gene or mRNA of either the virus or the host genome. Diagnostic microarrays are dependent for their utility and versatility on generic, multiplex or random polymerase chain reactions that will amplify any of several (unknown) viral target sequences from a patient sample. In this review, the existing and potential applications of microarrays in virology, and the problems that need to be overcome for future success, are discussed.