Alternative detection of SARS-CoV-2 RNA by a new assay based on mass spectrometry.

OBJECTIVES: Accurate detection of SARS-CoV-2 RNA is essential to stopping the spread of SARS-CoV-2. The aim of this study was to evaluate the performance of the recently introduced MassARRAY® SARS-CoV-2 Panel and to compare it to the cobas® SARS-CoV-2 Test. METHODS: The MassARRAY® SARS-CoV-2 Panel consists of five assays targeting different sequences of the SARS-CoV-2 genome. Accuracy was determined using national and international proficiency panels including 27 samples. For clinical evaluation, 101 residual clinical samples were analyzed and results compared. Samples had been tested for SARS-CoV-2 RNA with the cobas® SARS-CoV-2 Test. RESULTS: When accuracy was tested with the MassARRAY® SARS-CoV-2 Panel, 25 of 27 (92.6%) samples revealed correct results. When clinical samples were analyzed with the MassARRAY® SARS-CoV-2 Panel and compared to the cobas® SARS-CoV-2 Test, 100 samples showed concordant results. One sample was found to be inconclusive with the MassARRAY® SARS-CoV-2 Panel. When time-to-results were compared, the new assay showed longer total and hands-on times. CONCLUSIONS: The MassARRAY® SARS-CoV-2 Panel showed a good performance and proved to be suitable for use in the routine diagnostic laboratory. Especially during phases of shortage of reagents and/or disposables, the new test system appears as beneficial alternative to standard assays used for detection of SARS-CoV-2 RNA.

Antiretroviral Treatment Simplification With 2-Drug Regimens: Impact of Transmitted Drug Resistance Mutations.

The frequency of clinically relevant transmitted drug resistance mutations (DRMs) against drugs used for 2-drug regimens was 15.6%, but only 2% were not eligible for 1 or more 2-drug regimens. More than 50% of patients harboring any clinically relevant DRMs were found to be part of genetic transmission clusters.

Prospective evaluation of three rapid molecular tests for seasonal influenza in patients presenting at an emergency unit.

BACKGROUND: For infection control measures, rapid accurate diagnostics on admission of patients with suspected seasonal influenza is crucial. OBJECTIVE: Prospective comparison of three rapid molecular tests for detection of influenza A/B RNA. STUDY DESIGN: Outpatients presenting at the Medical emergency department of Graz University Hospital with influenza-like illness and a requirement for hospitalization (n = 312) were studied. Nasopharyngeal swabs were collected with the 3 mL-version of the UTM™ Viral Transport Medium (Copan). Specimens were tested for influenza A and B RNA using the Alere™ i Influenza A & B (Abbott), the cobas® Influenza A/B (Roche), and the Xpert® Xpress Flu/RSV (Cepheid) tests. Results were compared to those obtained from the same specimen by the Influenza A/B R-GENE® (bioMerieux) test based on real-time PCR as reference method. RESULTS: Overall sensitivities of the Abbott, Roche, and Cepheid tests were 90.5%, 96.0%, and 97.0%, overall specificities 99.4%, 97.6%, and 98.2% respectively. With the Abbott and the Cepheid tests, all specimens gave valid results, while the Roche test showed invalid results in 37 (12.1%) specimens. Total time to result for the Abbott, Roche, and Cepheid tests was 18 min, 22 min, and 32 min respectively. CONCLUSIONS: The Abbott test lacked sensitivity, the Roche test was impaired by a high number of invalid results. Overall, despite the longest total time to result, the Cepheid test showed the best performance to detect influenza virus RNA in symptomatic patients presenting at an emergency unit in this study.

First identification of a recombinant form of hepatitis C virus in Austrian patients by full-genome next generation sequencing.

Hepatitis C virus (HCV) intergenotypic recombinant forms have been reported for various HCV genotypes/subtypes in several countries worldwide. In a recent study, four patients living in Austria had been identified to be possibly infected with a recombinant HCV strain. To clarify results and determine the point of recombination, full-genome next-generation sequencing using the Illumina MiSeq v2 300 cycle kit (Illumina, San Diego, CA, USA) was performed in the present study. Samples of all of the patients contained the recombinant HCV strain 2k/1b. The point of recombination was found to be within the HCV NS2 gene between nucleotide positions 3189-3200 based on H77 numbering. While three of four patients were male and had migration background from Chechnya (n = 2) and Azerbaijan (n = 1), the forth patient was a female born in Austria. Three of the four patients including the female had intravenous drug abuse as a risk factor for HCV transmission. While sequencing techniques are limited to a few specialized laboratories, a genotyping assay that uses both ends of the HCV genome should be employed to identify patients infected with a recombinant HCV strain. The correct identification of recombinant strains also has an impact considering the tailored choice of anti-HCV treatment.

Evaluation of the new cobas® HCV genotyping test based on real-time PCRs of three different HCV genome regions.

BACKGROUND: Determination of the hepatitis C virus (HCV) genotype and discrimination between HCV subtypes 1a and 1b is still mandatory prior to anti-HCV treatment initiation. The aim of this study was to evaluate the performance of the recently introduced cobas® HCV GT assay (Roche) and to compare it to two comparator assays. METHODS: The cobas® HCV GT assay is based on primer-specific real-time polymerase chain reaction (PCR). For comparison, the TRUGENE® HCV 5'NC Genotyping Kit (Siemens) and the VERSANT® HCV Genotype 2.0 Assay (Siemens) were employed. Accuracy of the new assay was determined using proficiency panels. For clinical evaluation, 183 residual clinical samples obtained from patients with chronic hepatitis C infection were included. RESULTS: When accuracy was tested, panel members containing HCV subtypes 1a, 1b, and 3a were identified as expected; however, the new assay failed to identify low titer panel members containing HCV subtype 5a correctly. Of 183 clinical samples, 160 gave concordant results. For seven samples, an indeterminate result was reported with the cobas® HCV GT assay and the remaining 16 samples were found discordant with one of the comparator assays. When time-to-results of the assays were compared, the new assay showed shorter total time and similar hands-on time per sample. CONCLUSIONS: The cobas® HCV GT assay showed a good performance and proved to be suitable for use in the routine diagnostic laboratory. Due to the high level of automation, fast and reliable results are obtained with short hands-on time.

Analytical and clinical performance of a new molecular assay for Epstein-Barr virus DNA quantitation.

Quantitation of EBV DNA has been shown to be a useful tool to identify and monitor patients with immunosuppression and high risk for EBV-associated disease. In this study, the analytical and clinical performance of the new Realquality RS-EBV Kit (AB Analitica, Padova, Italy) was investigated. The clinical performance was compared to that of the EBV R-gene (bioMerieux, Varilhes, France) assay. When the accuracy of the new assay was tested, all results except of one were found to be within ±0.5log10 unit of the expected panel results. Determination of linearity showed a quasilinear curve, the between day imprecision ranged from 18% to 88% and the within run imprecision from 16% to 53%. When 96 clinical EDTA whole blood samples were tested, 77 concordant and 19 discordant results were obtained. When the results for the 69 samples quantifiable with both assays were compared, the new assay revealed a mean 0.31log10 unit higher measurement. The new assay proved to be suitable for the detection and quantitation of EBV DNA in EDTA whole blood in the routine diagnostic laboratory. The variation between quantitative results obtained by the assays used in this study reinforces the use of calibrators traceable to the existing international WHO standard making different assays better comparable.

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

BACKGROUND: Accurate quantitation of hepatitis C virus (HCV) RNA is mandatory for the management of anti-HCV therapy. OBJECTIVES: The 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. STUDY DESIGN: The molecular assays for quantitation of HCV RNA were performed according to the manufacturer's package insert instructions. HCV genotypes/subtypes/isolates, and mutations in the 5'NCR were detected by direct sequencing. RESULTS: When 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. CONCLUSION: The 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.

Evaluation of four molecular assays for detection of pandemic influenza A (H1N1) 2009 virus in the routine diagnostic laboratory.

BACKGROUND: Detection and differentiation of influenza A viral RNA and influenza A (H1N1) 2009 viral RNA have gained significance because of their widespread community transmission. OBJECTIVE: To study the accuracy and the performance of four molecular assays for the detection and differentiation of influenza A viral RNA and influenza A (H1N1) 2009 viral RNA in the routine diagnostic laboratory. STUDY DESIGN: The accuracy of the molecular assays was determined with reference material. For evaluation of the performance, 104 clinical specimens were studied. Sample preparation was done on a fully automated extraction instrument. For amplification and detection of influenza RNA, all molecular assays evaluated were based on real-time PCR. RESULTS: When the accuracy was tested, the majority of assays yielded results as expected. When clinical samples were analyzed, 94 samples gave concordant results with all assays. One of the assays showed one false-negative result and another assay 10 false-negatives. CONCLUSION: The majority of assays evaluated in this study proved suitable for the detection and differentiation of influenza A viral RNA and influenza A (H1N1) 2009 viral RNA. All assays are easy to handle and provide results rapidly.

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

BACKGROUND AND OBJECTIVES: Stress 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. METHODS: A 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. RESULTS AND CONCLUSIONS: There 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.

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.

Evaluation of a novel standardized system for collection and quantification of oral fluid.

BACKGROUND: Standardization in collection and testing of oral fluid is lacking. METHODS: A novel standardized collection and quantification system for oral fluid testing was evaluated. Sample collection volumes were determined. For determination of the saliva content in oral fluid samples, tartrazine, which is an integral part of the saliva extraction solution serving as an internal standard, was used. Results were compared with those obtained by employing glucose as a 'supplemental internal standard' for determination. Analytical performance of the new system for collection and quantification of oral fluid was evaluated. Calcium and magnesium analyte concentrations in oral fluid samples were measured with a routine laboratory method and compared to a reference method. RESULTS: Volumes of oral fluid samples collected ranged from 4.9 to 10.5 mL. The mean saliva content in oral fluid samples was found to be 65.5 percent by volume (vol.-%) when determined through the tartrazine concentration and 65.0 vol.-% when determined through the glucose concentration. Evaluation of analytical performance revealed interassay imprecision coefficients of variation (CVs) ranging from 1.5% to 3.2% and intraassay imprecision CVs from 1.1% to 2.5%. When linearity was tested, a quasi-linear curve was observed (R2=0.99). Comparison of two different methods for determination of calcium and magnesium concentrations showed correlations of R2=0.96 for calcium and R2=0.97 for magnesium. CONCLUSIONS: The new system for collection and quantification of oral fluid helps to improve standardization of pre-analytics in oral fluid testing and to provide reliable and accurate quantification of analytes in oral fluid samples.

Cyclooxygenase inhibition in human monocytes increases endotoxin-induced TNF alpha without affecting cyclooxygenase-2 expression.

Human endotoxin-stimulated adherent monocytes were used in order to determine whether or not NSAIDs influence cyclooxygenase-2 and/or tumor necrosis factor (TNF)alpha expression within the range of inhibitor concentrations that are required to suppress prostaglandin biosynthesis. Exogenous prostaglandin E(2) (IC(50)<5 nM) inhibited endotoxin-induced TNFalpha mRNA and protein while, up to 1 microM, it did not significantly affect cyclooxygenase-2 mRNA expression. Similar results were obtained using the membrane-permeable cAMP analogue db-cAMP, which caused preferential inhibition of TNFalpha expression. Indomethacin or lysine-acetylsalicylic acid concentration-dependently inhibited prostaglandin E(2) biosynthesis and, at concentrations causing near-complete inhibition, enhanced TNFalpha mRNA and protein expression without significantly influencing cyclooxygenase-2 mRNA. In addition, by facilitating endotoxin-induced TNFalpha expression, indomethacin or lysine-acetylsalicylic acid counteracted dexamethasone-induced inhibition of TNFalpha biosynthesis, thereby exhibiting an effect opposite to that of exogenous prostaglandin E(2). The results suggest that in human endotoxin-stimulated monocytes, NSAIDs can enhance TNFalpha expression through inhibition of cyclooxygenase and the resulting decrease in prostanoid biosynthesis.

Fully automated detection of hepatitis C virus RNA in serum and whole-blood samples.

In this study, we established a fully automated molecular assay for qualitative detection of hepatitis C virus (HCV) in serum and whole-blood samples and compared it with conventional molecular assays, including manual HCV RNA extraction protocols. Whole-blood samples were collected from patients with and without chronic HCV infection in EDTA tubes and nucleic acid stabilization tubes (NASTs). Prior to HCV RNA extraction, the HCV Internal Control (IC), derived from the COBAS AMPLICOR HCV test, version 2.0 (Roche Molecular Diagnostics), was added. The new assay was based on an automated extraction protocol on the MagNA Pure LC instrument (Roche Applied Science), followed by automated reverse transcription, amplification, hybridization, and detection on the Cobas Amplicor analyzer (Roche Molecular Diagnostics). The detection limit of the new assay was found to be similar to those of conventional molecular assays. In clinical samples, 100% agreement between the new assay and conventional methods was observed. The introduced amount of IC was detected in 45 of 45 serum samples, 41 of 45 EDTA tube whole-blood samples, and 43 of 45 NAST whole-blood samples. Retesting led to more frequent IC detection. The fully automated molecular assay was found to be suitable for detection of HCV RNA in different kinds of sample materials. It may be recommended for use in the high-throughput routine molecular diagnostic laboratory.

Rapid detection of enterovirus infection by automated RNA extraction and real-time fluorescence PCR.

BACKGROUND: Molecular detection has been shown to be superior to tissue culture for the detection of enteroviruses in cerebrospinal fluid (CSF) specimens. OBJECTIVES: In this study, a qualitative molecular assay based on automated RNA extraction with the MagNA Pure LC and real-time PCR on the LightCycler (LC) instrument was evaluated and compared with an in-house molecular assay. STUDY DESIGN: A total of 109 CSF specimens were investigated for the comparative study. The detection limit of the new molecular assay was determined with 10-fold dilutions of two enterovirus strains and with the Third European Union Concerted Action Enterovirus Proficiency Panel. RESULTS: With the enterovirus strains, the detection limit of the LC assay was found to be 0.1 TCID(50) (50% tissue culture infective dose). When samples of the Third European Union Concerted Action Enterovirus Proficiency Panel were tested, both molecular assays gave identical results to the expected results, which were based upon the results of three reference laboratories using a total of four different molecular methods before distribution of the panel. When clinical specimens were tested, there was a correlation between the LC assay and the in-house assay in 105 of 109 cerebrospinal fluids. CONCLUSIONS: The new molecular assay allows rapid detection of enterovirus RNA in CSF. It was found to be labor saving and showed sufficient sensitivity.

Evaluation of an automated sample preparation protocol for quantitative detection of hepatitis C virus RNA.

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.