Lack of sensitivity of an IVD/CE-labelled kit targeting the S gene for detection of SARS-CoV-2.

OBJECTIVES: New molecular tests for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are being rapidly launched in response to the coronavirus disease 2019 (COVID-19) pandemic. The aim of this study was to evaluate the analytical and clinical performance of the VIASURE SARS-CoV-2 S gene RT-PCR Kit on the BD Max™ system and to compare results with those obtained with the cobas® SARS-CoV-2 test on the cobas® 6800 system. METHODS: For testing the analytical performance, reference material was used. Clinical samples (n = 101) obtained from individuals with symptoms compatible with COVID-19 were studied. Oropharyngeal and nasopharyngeal swabs were collected by using either ESwab™ or UTM™ collection systems. RESULTS: When the analytical performance was evaluated, the sample containing the lowest SARS-CoV-2 concentration tested negative with the VIASURE test whereas results obtained with the cobas® test were found to be concordant with the results expected. Six out of the 101 clinical samples (5.9%) showed an inhibition with the VIASURE test. When analysing the remaining 95 clinical samples, 27 were found to be negative with both assays. Of 68 samples that were positive with the cobas® test, the VIASURE test missed 21 (30.9 %) samples. All of those 21 samples had shown Ct values ≥ 31 with the cobas® 6800 system. None of the samples tested positive with the VIASURE test and negative with the cobas® test. CONCLUSIONS: The VIASURE test was impaired by a lack of sensitivity and a relatively high number of invalid results. When using the VIASURE test for routine testing, a significant number of COVID-19-positive samples would have been missed.

Successful antibiotic treatment of Borreliosis associated pseudolymphomatous systemic infiltrates.

The clinical management of late stage Borreliosis can be difficult due to various associated symptoms and signs and cumbersome microbiological tests. We report a case of successful antibiotic treatment of Borreliosis-associated pseudolymphomatous infiltrates in bone marrow and lymph nodes, which were diagnosed by bone marrow trephine biopsy and positron emission tomography.

Determination of human immunodeficiency virus type 1 subtypes by a rapid method useful for the routine diagnostic laboratory.

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.

Effects of storage and type of blood collection tubes on hepatitis C virus level in whole blood samples.

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.

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.

Detection of Herpes simplex virus DNA by real-time PCR.

Molecular detection of herpes simplex virus (HSV) DNA is recognized as the reference standard assay method for the sensitive and specific diagnosis of central nervous system infections caused by HSV. In this study, a molecular assay based on real-time PCR on the LightCycler (LC) instrument was evaluated and compared with a home-brew molecular assay. The detection limit of the LC assay was determined with 10-fold dilutions of plasmid pS4 with the SalI restriction fragment of the DNA polymerase gene and with the First European Union Concerted Action HSV Proficiency Panel. A total of 59 cerebrospinal fluid (CSF) specimens were investigated for the comparative study. With plasmid pS4, the detection limit of the LC assay was found to be 10(4) copies per ml, i.e., 12.5 copies per run. When samples of the First European Union Concerted Action HSV Proficiency Panel were tested, 2x10(3) to 5x10(3) HSV type 1 genome equivalents (GE) per ml, i.e., 2.5 to 6.3 GE per run, could consistently be detected. There was a correlation between the LC assay and the home-brew assay in 55 of 59 specimens. In conclusion, the LC assay allows very rapid detection of HSV DNA in CSF. It was found to be laborsaving and showed sufficient sensitivity.

Identification of different states of hepatitis B virus infection with a quantitative PCR assay.

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 x 10(8) copies/ml) was significantly higher than that for patients after HBeAg seroconversion (6.2 x 10(3) copies/ml) and than that for inactive HBsAg carriers (5.6 x 10(3) 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 AMPLILINK software for the COBAS AMPLICOR system.

The use of AMPLILINK version 1.0 software was evaluated for the operation and control of one COBAS AMPLICOR instrument and for two COBAS AMPLICOR instruments run simultaneously to perform and detect nucleic acid amplification reactions. A total of 3,384 results were analyzed. The initial accuracy of the results was 99.91%. Three errors of omission of transfer of data from the COBAS AMPLICOR to the AMPLILINK system were observed. Two of these errors were from a single specimen, where both the analyte and internal control results were not transmitted. These errors did not interfere with the correctness of any other data. There were no interruptions of runs, and no data were mixed. AMPLILINK increased convenience, saved labor, and was found to be a very useful addition for clinical laboratories performing molecular-diagnostic procedures with the COBAS AMPLICOR system.

Automation of polymerase chain reaction-based systems for detection of hepatitis C virus RNA.

Polymerase chain reaction-based molecular assays are gaining increasing importance in the diagnosis and monitoring of infectious diseases. Over the past several years, the development and application of these techniques has initiated a revolution in the diagnosis and monitoring of hepatitis C infection. Presently, molecular assays are exclusively done in especially dedicated laboratories. Advances in automation will bring these technologies into routine diagnostic laboratories and will make them widely used.

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

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.

Evaluation of a new assay for HBV DNA quantitation in patients with chronic hepatitis B.

BACKGROUND: The Amplicor HBV Monitor Test for quantitative determination 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. OBJECTIVE: The performance of the Amplicor HBV Monitor Test was evaluated in a routine diagnostic laboratory. The Amplicor HBV Monoitor assay was compared to the Digene Hybrid Capture System HBV DNA assay for the quantitation of HBV in patient sera. STUDY DESIGN: Sensitivity and reproducibility were determined with 10-fold dilution series of two Eurohep HBV reference plasma specimens. Furthermore, 196 sera from 14 children with chronic HBV infection and interferon therapy were tested with both assays. RESULTS: The detection limit was found to be 10(3) copies/ml with the Amplicor PCR assay compared to 10(6) to 10(7) copies/ml with the Digene hybridization assay. Both assays were quasi-linear over the measurable ranges. The new PCR assay proved to be very reliable. With the Amplicor PCR assay, 26.2% of the HBV DNA-positive clinical samples were found between 10(3) and 10(7) copies/ml and all of them tested below the detection limit with the hybridization assay. CONCLUSION: The Amplicor HBV Monitor Test shows excellent sensitivity and provides a valuable tool for the detection of HBV DNA in serum. It can be used for recognizing those patients who might benefit from antiviral therapy, for evaluation of the efficacy of anti-HBV therapy, and for validation of blood products.

Evaluation of molecular parameters for routine assessment of viremia in patients with chronic hepatitis C who are undergoing antiviral therapy.

OBJECTIVE: To define the usefulness of molecular parameters in patients with chronic hepatitis C who are undergoing antiviral therapy. Anti-hepatitis C virus (HCV) treatment was monitored by determination of serum HCV load and by presence of HCV RNA in peripheral blood mononuclear cells (PBMCs). STUDY DESIGN/METHODS: Fifty-one patients with chronic hepatitis C undergoing antiviral therapy with interferon-alpha plus ribavirin were studied. Serum HCV RNA load was tested with a quantitative assay (Amplicor HCV Monitor Test) before, during, and up to 12 months after end of treatment. If HCV RNA was not detectable, serum samples were subsequently tested with a qualitative assay (Cobas Amplicor HCV Test) and corresponding ethylenediaminetetraacetic acid (EDTA)-treated blood was checked for presence of HCV RNA in peripheral blood mononuclear cells (PBMCs). Sustained virologic response was defined by loss of HCV RNA 12 months after the end of treatment. RESULTS: Four patients (7.8%) were found to be sustained virologic responders, 17 (33.3%) were transient virologic responders, and 30 (58.8%) were virologic nonresponders. No significant difference was found in the median pretreatment serum HCV RNA load between sustained virologic responders, transient virologic responders, and virologic nonresponders. At 1 month after start of therapy, HCV RNA was not detectable with both the serum and the PBMC assay in 12 (23.5%) of 51 patients. Four remained HCV RNA-negative until 12 months after the end of treatment. In 14 of 17 transient virologic responders, reappearance of HCV RNA was detected earlier in PBMCs than in serum. CONCLUSIONS: Based on these results in 51 patients, quantitation of baseline serum HCV RNA does not appear to be a decisive factor to the management of the individual patient. Early assessment of serum HCV RNA level after start of anti-viral treatment seems to be of major importance to identify virologic nonresponders. Reappearance of HCV RNA may be demonstrated earlier in PBMCs than in serum.

Performance of the automated COBAS AMPLICOR system for the detection of hepatitis C virus RNA.

BACKGROUND: The COBAS AMPLICOR (CA) instrument for the amplification and detection steps of the AMPLICOR molecular diagnostic assays has recently been introduced. The system contains a single thermal cycler with two independently controlled heating/cooling blocks, a pipettor, a magnetic particle washer, a photometer and an incubator. OBJECTIVE: The performance of the CA instrument was evaluated in a routine diagnostic laboratory for the detection of hepatitis C virus (HCV) RNA. The new system was compared with the corresponding microwell plate assay (AMPLICOR HCV Test). STUDY DESIGN: Routine clinical sera (350) from hemodialysis patients and patients with chronic HCV infection and interferon therapy were studied. If discrepant results were obtained, both assays were repeated (specimen preparation, amplification and detection); in addition, the HCV copy number was determined with the AMPLICOR HCV MONITOR Test. RESULTS: There was a correlation between the CA HCV Test and the AMPLICOR HCV Test in 341 of 350 specimens (97%). After resolution of 9 discrepant results, the CA HCV Test gave a sensitivity of 97.8% and a specificity of 99.4%. The most common reason for discrepant results was a low HCV RNA copy number. CONCLUSION: The CA system was found to be a labor-saving, fast and reliable instrument for the amplification and detection steps of a RT-PCR molecular assay for detection of HCV RNA.

Quantitation and genotyping of hepatitis C virus RNA in sera of hemodialysis and AIDS patients.

BACKGROUND: Hepatitis C virus (HCV) infection is highly prevalent in hemodialysis and AIDS patients. Little information exists about the viral load in those patients. OBJECTIVE: To characterize HCV infection in hemodialysis and AIDS patients, the viral load in the sera was measured. Results were compared with genotypes, gender of the patients, and biochemical markers of active hepatitis. STUDY DESIGN: Sera from a total of 442 patients were screened with a third-generation EIA, and anti-HCV immunoreactivity was confirmed with the Wellcozyme HCV Western Blot. After qualitative PCR with the Amplicor PCR Test, positives were genotyped using a reverse hybridization test. Determination of HCV levels was done with the Amplicor HCV Monitor assay. RESULTS: HCV RNA was detected in the sera of 95 (74.8%) EIA-positive patients. HCV RNA levels ranged from 1 x 10(4) to 1.4 x 10(6) molecules of HCV RNA/ml. Median HCV RNA levels of AIDS patients were slightly higher than those of hemodialysis patients. Male patients had higher median HCV RNA levels compared with female patients. No association between HCV RNA levels and both alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels was found. The most common genotypes were type 1b and type 1a, followed by type 3, type 4, and type 2a. There were no significant differences in HCV RNA levels among patients with genotypes 1a, 1b, and 2a. Patients infected with types 3 and 4, respectively, had significantly lower HCV RNA levels compared with other genotypes. CONCLUSION: Because the Amplicor HCV Monitor assay allows quantitation of low-titer viremic patients, HCV RNA levels were distinctly lower compared with previous reports. HCV RNA levels of males did not differ significantly from those of females. ALT and AST are very poor indicators of ongoing HCV infection. Patients with chronic type 3 or type 4 HCV infection tended to have lower HCV RNA levels.