Ultrasensitive amplification refractory mutation system real-time PCR (ARMS RT-PCR) assay for detection of minority hepatitis B virus-resistant strains in the era of personalized medicine.

Resistance to antiviral treatment for chronic hepatitis B virus (HBV) has been associated with mutations in the HBV polymerase region. This study aimed at developing an ultrasensitive method for quantifying viral populations with all major HBV resistance-associated mutations, combining the amplification refractory mutation system real-time PCR (ARMS RT-PCR) with a molecular beacon using a LightCycler. The discriminatory ability of this method, the ARMS RT-PCR with molecular beacon assay, was 0.01 to 0.25% for the different HBV resistance-associated mutations, as determined by laboratory-synthesized wild-type (WT) and mutant (Mut) target sequences. The assay showed 100% sensitivity for the detection of mutant variants A181V, T184A, and N236T in samples from 41 chronically HBV-infected patients under antiviral therapy who had developed resistance-associated mutations detected by direct PCR Sanger sequencing. The ratio of mutant to wild-type viral populations (the Mut/WT ratio) was >1% in 38 (63.3%) of 60 samples from chronically HBV-infected nucleos(t)ide analogue-naive patients; combinations of mutations were also detected in half of these samples. The ARMS RT-PCR with molecular beacon assay achieved high sensitivity and discriminatory ability compared to the gold standard of direct PCR Sanger sequencing in identifying resistant viral populations in chronically HBV-infected patients receiving antiviral therapy. Apart from the dominant clones, other quasispecies were also quantified. In samples from chronically HBV-infected nucleos(t)ide analogue-naive patients, the assay proved to be a useful tool in detecting minor variant populations before the initiation of the treatment. These observations need further evaluation with prospective studies before they can be implemented in daily practice.

Comparative evaluation of the performance of the Abbott RealTime HIV-1 assay for measurement of HIV-1 plasma viral load on genetically diverse samples from Greece.

BACKGROUND: HIV-1 is characterized by increased genetic heterogeneity which tends to hinder the reliability of detection and accuracy of HIV-1 RNA quantitation assays. METHODS: In this study, the Abbott RealTime HIV-1 (Abbott RealTime) assay was compared to the Roche Cobas TaqMan HIV-1 (Cobas TaqMan) and the Siemens Versant HIV-1 RNA 3.0 (bDNA 3.0) assays, using clinical samples of various viral load levels and subtypes from Greece, where the recent epidemiology of HIV-1 infection has been characterized by increasing genetic diversity and a marked increase in subtype A genetic strains among newly diagnosed infections. RESULTS: A high correlation was observed between the quantitative results obtained by the Abbott RealTime and the Cobas TaqMan assays. Viral load values quantified by the Abbott RealTime were on average lower than those obtained by the Cobas TaqMan, with a mean (SD) difference of -0.206 (0.298) log(10) copies/ml. The mean differences according to HIV-1 subtypes between the two techniques for samples of subtype A, B, and non-A/non-B were 0.089, -0.262, and -0.298 log(10) copies/ml, respectively. Overall, differences were less than 0.5 log(10) for 85% of the samples, and >1 log(10) in only one subtype B sample. Similarly, Abbott RealTime and bDNA 3.0 assays yielded a very good correlation of quantitative results, whereas viral load values assessed by the Abbott RealTime were on average higher (mean (SD) difference: 0.160 (0.287) log(10) copies/ml). The mean differences according to HIV-1 subtypes between the two techniques for subtype A, B and non-A/non-B samples were 0.438, 0.105 and 0.191 log(10) copies/ml, respectively. Overall, the majority of samples (86%) differed by less than 0.5 log(10), while none of the samples showed a deviation of more than 1.0 log(10). CONCLUSIONS: In an area of changing HIV-1 subtype pattern, the Abbott RealTime assay showed a high correlation and good agreement of results when compared both to the Cobas TaqMan and bDNA 3.0 assays, for all HIV-1 subtypes tested. All three assays could determine viral load from samples of different HIV-1 subtypes adequately. However, assay variation should be taken into account when viral load monitoring of the same individual is assessed by different systems.

Development of a new ultra sensitive real-time PCR assay (ultra sensitive RTQ-PCR) for the quantification of HBV-DNA.

BACKGROUND: Improved sensitivity of HBV-DNA tests is of critical importance for the management of HBV infection. Our aim was to develop and assess a new ultra sensitive in-house real-time PCR assay for HBV-DNA quantification (ultra sensitive RTQ-PCR). RESULTS: Previously used HBV-DNA standards were calibrated against the WHO 1st International Standard for HBV-DNA (OptiQuant(R) HBV-DNA Quantification Panel, Accrometrix Europe B.V.). The 95% and 50% HBV-DNA detection end-point of the assay were 22.2 and 8.4 IU/mL. According to the calibration results, 1 IU/mL equals 2.8 copies/mL. Importantly the clinical performance of the ultra sensitive real-time PCR was tested similar (67%) to the Procleix Ultrio discriminatory HBV test (dHBV) (70%) in low-titer samples from patients with occult Hepatitis B. Finally, in the comparison of ultra sensitive RTQ-PCR with the commercially available COBAS TaqMan HBV Test, the in-house assay identified 94.7% of the 94 specimens as positive versus 90.4% identified by TaqMan, while the quantitative results that were positive by both assay were strongly correlated (r = 0.979). CONCLUSIONS: We report a new ultra sensitive real time PCR molecular beacon based assay with remarkable analytical and clinical sensitivity, calibrated against the WHO 1st International standard.

Molecular characterization of occult hepatitis B cases in Greek blood donors.

The use of sensitive nucleic acid testing for hepatitis B virus in blood donors revealed a number of HBV DNA(+) cases among HBsAg(-) donors, a status known as occult HBV infection. The purpose of this study was the serological and molecular characterization of occult HBV infection in Greek blood donors. A prospective study was undertaken in order to identify occult HBV infection cases in blood donors. As part of the routine screening of blood donations in Greece, blood units were screened individually by a multiplex HIV-1/HCV/HBV nucleic acid assay. Initially reactive samples were retested with discriminatory assays. HBV DNA(+)/HBsAg(-) samples were tested further for HBV serological markers and HBV DNA was quantified by real-time PCR. Molecular characterization was performed by sequencing the envelope and polymerase genes of HBV. Preliminary screening revealed 21 occult cases with the following patterns: anti-HBc only: 7 donors, anti-HBc/anti-HBs: 7 donors, anti-HBc/anti-HBe: 5 donors, anti-HBc/anti-HBs/anti-HBe: 2 donors. In all cases, the HBV DNA load was <351 IU/ml. Sequencing was successful in 10 donors (classified within genotype D) revealing several amino acid substitutions related to diagnostic escape and antiviral resistance. HBsAg diagnostic failure and low viral replication in occult HBV infection carriers could possibly be attributed to multiple changes in envelope and polymerase regions, respectively.

HBV viremia in newborns of HBsAg(+) predominantly Caucasian HBeAg(-) mothers.

BACKGROUND: Hepatitis B virus infection is an important public health problem worldwide and eliminating mother-to-infant transmission is important to decrease the prevalence of chronic HBV-infection. Although, immunoprophylaxis given at birth largely prevents mother-to-infant transmission, perinatal HBV viremia has been reported in HBsAg(-) newborns born mainly to HBeAg(+) women in endemic areas. OBJECTIVES: To examine the incidence of perinatal HBV viremia in newborns of HBsAg(+) predominantly HBeAg(-) mothers. STUDY DESIGN: Peripheral blood was obtained at birth from 109 HBsAg(+) mothers and their newborns before the administration of active-passive immunoprophylaxis. Infants were prospectively followed and appropriately vaccinated. RESULTS: Although most (92.7%) of the HBsAg(+) mothers were HBeAg(-), 73.4% had detectable HBV viremia. Neonatal viremia was detected in 3/8 (37.5%) and 24/101 (23.8%) newborns of HBeAg(+) and HBeAg(-) mothers, respectively (p=0.386). However, HBV-DNA levels were significantly higher in newborns of HBeAg(+) mothers (p=0.025). No child developed chronic HBV infection, but one child had evidence of subclinical hepatitis. CONCLUSIONS: Although the clinical significance of low viremia levels in almost one in four newborns of HBsAg(+) mothers in a low endemicity area is unclear, it may enhance our understanding of HBV mother-to-infant transmission.

HBV virological assessment.

Management of hepatitis B virus (HBV) infected patients involves serological diagnosis, quantitation of HBV-DNA and measurement of HBV drug resistance. Different serological markers such as HBsAg, anti-HBs, anti-HBc (total and IgM), HBeAg and anti-HBe are assessed by immunoassays in order to define the infection status. The emergence of surface mutants however is a continuous challenge to design more effective immunoassays. Commercially available quantitative HBV-DNA assays with increased sensitivity and wider linear range give a more accurate estimate of viral replication and contribute decisively in the initiation and the monitoring of the response to HBV therapy. Genotypic drug resistance assays are important diagnostic tools, since the administration of nucleoside/nucleotide analogues to HBV infected patients leads to the development of drug resistance patterns very much dependent on the treatment regimen. Special issues have to be taken into consideration regarding HBV/HIV-1 co-infected patients, since concominant HIV and HBV replication results in higher rates of HBV replication. Current efforts are focused on the standardization of HBV-DNA assays (qualitative and quantitative), of HBV drug resistance assays as well as in the development of new assays and markers that will help in the prognosis and management of HBV infection (quantitative detection of pre-core mutants and HBV ccc-DNA assays).