Brigitte I. Santner

Identification of Different States of Hepatitis B Virus Infection with a Quantitative PCR Assay

ABSTRACT The level of hepatitis B virus (HBV) DNA in serum reflects the replicative activity of HBV. To compare serum HBV DNA levels in different states of hepatitis B, 47 sera of patients with HBeAg-positive chronic hepatitis B, 4 sera of patients with HBeAg-negative chronic hepatitis B, 40 samples of patients after HBeAg seroconversion during alpha interferon treatment, 57 sera of inactive HBsAg carriers, and 42 sera of patients who had recovered from chronic hepatitis B more than 12 months prior to blood collection were checked for the presence of HBV DNA with the Amplicor HBV Monitor Test. In patients with HBeAg-positive chronic hepatitis B, the median of serum HBV DNA levels (8.3 × 108 copies/ml) was significantly higher than that for patients after HBeAg seroconversion (6.2 × 103 copies/ml) and than that for inactive HBsAg carriers (5.6 × 103 copies/ml). None of the patients who had recovered from hepatitis B had detectable HBV DNA in serum. Quantitative PCR proved to be a valuable tool for identification of different states of HBV infection. This technique was found to be a good method for determination of serum HBV DNA levels both for patients with HBeAg seroconversion and for inactive carriers who showed low viremia not detectable by conventional hybridization assays.

Evaluation of an Automated Sample Preparation Protocol for Quantitative Detection of Hepatitis C Virus RNA

ABSTRACT The COBAS AMPLIPREP instrument for automated sample preparation has recently been introduced. In this study, the COBAS AMPLIPREP/COBAS AMPLICOR HCV MONITOR test, which includes this new molecular device, was evaluated and compared to the COBAS AMPLICOR HCV MONITOR test, which includes a manual extraction protocol. Interassay and intra-assay variation, precision, and linearity were determined, and a total of 130 clinical specimens were investigated. For determination of interassay variation, coefficients of variation were found to be between 9 and 59% for the COBAS AMPLIPREP/COBAS AMPLICOR HCV MONITOR test and between 13 and 69% for the COBAS AMPLICOR HCV MONITOR test. For determination of intra-assay variation, coefficients of variation were found to be between 7 and 13% for the COBAS AMPLIPREP/COBAS AMPLICOR HCV MONITOR test and between 8 and 16% for the COBAS AMPLICOR HCV MONITOR test. When precision of the COBAS AMPLIPREP/COBAS AMPLICOR HCV MONITOR test was tested, all results were found to be within ±0.5 log of the expected results. Determination of linearity resulted in a quasilinear curve over 3 logs. When clinical samples were tested with the COBAS AMPLIPREP/COBAS AMPLICOR HCV MONITOR test and compared with the COBAS AMPLICOR HCV MONITOR test, all results were found within ±0.5 log. In conclusion, the assay, which included the new molecular device, proved to be suitable for the routine molecular laboratory. It was found to be laborsaving and easy to handle.

Feasibility of testing three salivary stress biomarkers in relation to naturalistic traffic noise exposure.

BACKGROUND AND OBJECTIVESnStress dependent alterations of the salivary biomarkers alpha-amylase (sAA), salivary chromogranin A (sCgA) and salivary cortisol (sC) have been reported in numerous studies recently. The aim of this pilot study was to investigate the feasibility of testing sAA, sCgA and sC in relation to naturalistic traffic noise exposure in order to monitor a direct stress response in a laboratory setup.nnnMETHODSnA total of twenty study participants were exposed to binaurally recorded naturalistic traffic noise samples containing 75 dB (L(A,)eq) for 20 minutes via a loudspeaker system. Saliva was collected directly before and after defined exposure to naturalistic traffic noise. Determination of sAA was performed enzymatically on a Hitachi 912 laboratory analyzer, sCgA was determined by ELISA technique and sC was determined using a RIA assay.nnnRESULTS AND CONCLUSIONSnThere was a significant increase of sAA and sC concentrations after traffic noise exposure (p=0.045; p=0.01), whereas for sCgA this was not observed (p=0.48). Measuring of sAA and sC appear to be feasible to investigate direct stress effects in relation to naturalistic traffic noise exposure in a laboratory setup. Considering the small sample size of this pilot study, these observations need to be further proved in a larger explorative study.

Effects of Storage and Type of Blood Collection Tubes on Hepatitis C Virus Level in Whole Blood Samples

ABSTRACT In this study, we compared serum hepatitis C virus (HCV) RNA concentrations with HCV RNA concentrations in whole blood collection tubes, including two different types of EDTA tubes and nucleic acid stabilization tubes (NASTs). We also investigated the impact of a processing delay on HCV RNA concentration in these tubes. In NASTs, the mean HCV RNA concentration was comparable to the mean serum HCV RNA concentration at “date zero.” In EDTA tubes, mean baseline HCV RNA concentrations were higher. Storage at room temperature up to 96 h did not result in a decline of HCV RNA concentration in any of the whole blood collection tubes. In NASTs, HCV RNA concentrations remained stable during the whole study period, whereas a significant increase of HCV RNA was observed in both types of EDTA tubes at 96 h compared to date zero. We concluded that HCV RNA remains stable in NASTs at room temperature for at least 96 h, allowing greater flexibility in sample collection and transport.

Semiautomated Quantification of Hepatitis B Virus DNA in a Routine Diagnostic Laboratory

ABSTRACT The Cobas Amplicor HBV Monitor test for quantitative determination of hepatitis B virus (HBV) DNA in serum has recently been introduced. To evaluate the performance of this assay in a routine diagnostic laboratory, reproducibility of results was determined with the First European Union Concerted Action HBV Proficiency Panel and the Accurun 325 HBV DNA Positive Control, Series 300. Results for 270 routine serum samples were additionally evaluated. To avoid the retesting of a large number of samples due to titers exceeding the upper limit for the linear range of the assay, sera of patients with chronic hepatitis B (CHB) were diluted prior to the assay to 10−4 in normal human plasma, which is included in the assay. The mean coefficient of variation was 22.9% for all input HBV DNAs. Of 270 routine serum samples, 182 (150 sera from transplant donors and 32 sera from patients who had recovered from CHB) tested negative. Eighty-six sera were found to be HBV DNA positive; in six sera, HBV DNA levels were found to exceed the upper limit for the linear range of the assay and had to be retested. In the remaining two sera, inhibition occurred. The semiautomated Cobas Amplicor HBV Monitor test showed sufficient reproducibility and helped in avoiding human error. The relatively narrow linear range of detection is a limitation of the new assay.

Genotype impact on HCV RNA levels determined with the VERSANT HCV RNA 1.0 Assay (kPCR)

BACKGROUNDnAccurate quantitation of hepatitis C virus (HCV) RNA is mandatory for the management of anti-HCV therapy.nnnOBJECTIVESnThe genotype-dependent performance of the new commercially available VERSANT HCV RNA 1.0 Assay (kPCR) and the COBAS AmpliPrep/COBAS TaqMan HCV Quantitative Test, version 2.0 was investigated.nnnSTUDY DESIGNnThe molecular assays for quantitation of HCV RNA were performed according to the manufacturers package insert instructions. HCV genotypes/subtypes/isolates, and mutations in the 5NCR were detected by direct sequencing.nnnRESULTSnWhen members of a worldwide HCV performance panel including HCV subtypes 1a, 1b, 2a, 3b, and 4a were tested with the Siemens assay and the results were compared with those obtained by the Roche assay, the mean log10 unit differences for members containing HCV subtypes 1a, 1b, 3b, and 4a were found to be within ±0.5 log(10) units. For the panel member containing HCV subtype 2a, the HCV RNA concentration was found to be >0.5 log(10) units lower with the Siemens assay. When clinical samples were tested, the HCV RNA concentration of all samples containing HCV subtype 2a were found to be >0.5 log(10) units lower with the Siemens assay while that of certain HCV subtype 3a and 4a isolates were found to be >1.0 log(10) units lower.nnnCONCLUSIONnThe VERSANT HCV RNA 1.0 Assay substantially underestimates HCV RNA concentrations in HCV subtype 2a samples and in HCV subtype 3a and 4a samples containing certain isolates. This may be caused by mismatches with the target sequences due to the primer and/or probe design.

Reliable Detection and Quantitation of Viral Nucleic Acids in Oral Fluid : Liquid Phase-Based Sample Collection in Conjunction With Automated and Standardized Molecular Assays

Oral fluid has been used widely as sample matrix for the detection and quantitation of viral nucleic acids. However, in the vast majority of previous studies, various methods for collection of oral fluid and molecular assays lacking automation and standardization were used. In this study, a new standardized liquid phase‐based saliva collection system was employed followed by a fully automated viral nucleic acid extraction and real‐time PCR using commercially available in vitro diagnostics (IVD)/Conformité Européene (CE) labeled molecular assays. When the lower limit of detection of herpes simplex virus (HSV)‐1/2 DNA, varicella zoster virus (VZV) DNA, and hepatitis C virus (HCV) RNA in spiked oral fluid was tested, the results were found to be comparable to those with defined sample materials recommended by the assay manufacturers. When clinical specimens were investigated, 21 of 25 (84%) oral fluids obtained from patients with clinically apparent herpetic lesions tested positive for HSV DNA, 7 of 10 (70%) oral fluids obtained from patients with Ramsay Hunt Syndrome tested positive for VZV DNA, and 19 of 40 (48%) oral fluids collected from patients with chronic HCV infection tested positive for HCV RNA. The automated extraction instruments completed all extractions without malfunction and no inhibitions were observed throughout the entire study. Liquid phase‐based saliva collection in conjunction with automated and standardized commercially available molecular assays allows reliable quantitation of viral nucleic acids in oral fluid samples and may contribute to improved comparable and interpretable test results. J. Med. Virol. 80:1684–1688, 2008.

Determination of Human Immunodeficiency Virus Type 1 Subtypes by a Rapid Method Useful for the Routine Diagnostic Laboratory

ABSTRACT The existence of human immunodeficiency virus type 1 (HIV-1) subtypes has many important implications for the global evolution of HIV and for the evaluation of pathogenicity, transmissibility, and candidate HIV vaccines. The aim of this study was to establish a rapid method for determination of HIV-1 subtypes useful for a routine diagnostic laboratory and to investigate the distribution of HIV-1 subtypes in Austrian patients. Samples were tested by a subtyping method based on a 1.3-kb sequence of the polymerase gene generated by a commercially available drug resistance assay. The generated sequence was subtyped by means of an HIV sequence database. Results of 74 routine samples revealed subtype B (71.6%) as the predominant subtype, followed by subtype A (13.5%) and subtype C (6.8%). Subtypes E, F, G, and AE (CM240) were also detected. This subtyping method was found to be very easy to handle, rapid, and inexpensive and has proved suitable for high-throughput routine diagnostic laboratories. The specific polymerase gene sequence, however, must be existent.

Quantitative detection of hepatitis B virus DNA with a new PCR assay.

Abstract The Amplicor™ HBV Monitor Test for quantitative detection of serum hepatitis B virus (HBV) DNA has recently been introduced. This assay is based on PCR and a non-radioactive hybridization and detection system on microwell plates. Evaluation in a routine diagnostic laboratory showed excellent sensitivity and adequate reproducibility; however, a more automated format would be desirable. The Amplicor™ HBV Monitor Test is useful for recognizing those patients who might benefit from antiviral treatment and for evaluation of the efficacy of anti-hepatitis B virus treatment.

Detection of hepatitis C viral sequences in serum by ‘nested’ polymerase chain reaction (PCR) and a commercial single-round PCR assay

BACKGROUNDnDemonstration of the hepatitis C virus (HCV) genome is usually done with combined reverse transcription and polymerase chain reaction (RT-PCR) employing nested primer sets. Recently, a commercial PCR assay (Amplicor PCR assay), based on a simplified sample preparation procedure, a single, combined reverse transcription and polymerase chain reaction (RT-PCR), and a microwell plate capture and detection, has been developed.nnnOBJECTIVEnThe aim of the present study was to compare the new Amplicor assay with an in-house PCR. Additional testing included a third-generation enzyme immunoassay for anti-HCV antibodies, the Wellcozyme HCV Western Blot, which is equivalent to a third-generation recombinant immunoblot assay. Furthermore, HCV genotypes were classified.nnnSTUDY DESIGNnSera from a total of 127 patients were studied. After screening with a third-generation enzyme immunoassay (EIA), the Wellcozyme HCV Western Blot, was performed as well as the conventional RT-PCR and the Amplicor PCR. Specimens, which were found positive by testing with the Amplicor kit, were subjected to storage at room temperature for 96 h.nnnRESULTSnA total of 52 patients were found to be positive for anti-HCV by the third-generation EIA. With the Amplicor assay, the HCV genome was detected in 38 patients. In comparison with the in-house assay, two discrepant results were found. Resolution of discrepant samples increased the total number of true positives to 39. A good correlation was found between a positive anti-HCV test result and the presence of HCV-RNA by RT-PCR. No significant reduction in the amount of amplification product was observed by retesting of suboptimally stored samples with the Amplicor assay.nnnCONCLUSIONnBecause of the rapidity and the improved ease of handling, the Amplicor assay was found to be a good contribution for detection of HCV in serum.