Use of RT-PCR thermocycling program for the quantification of DNA viruses in a single run with RNA viruses: Example of Altona RealStar® HSV or VZV PCR kits.

Real-time PCR plays a key role in the diagnosis of viral infections. Multiple kits can detect or quantify genomes of various viruses with the same thermocycling program. Detection of RNA viruses includes an additional step of reverse transcription and challenge their detection in a single run with DNA viruses. We investigated the analytical performance of HSV-1, HSV-2 and VZV DNA quantification with Altona RealStar® PCR kits using the RT-PCR program for RNA viruses instead of the PCR program for DNA viruses. For each three viruses, Bland-Altman distribution did not show differences between both programs, and quantification curves generated with both thermocycling programs confirmed high correlation (R2 ≥ 0.9983). Detection of low viral load samples was evaluated, on 10-times repeat-test. All replicate samples were detected with both thermocycling programs and were quantified at similar viral loads (bias in log10 copies/mL: +0.05 (HSV-1), -0.01 (HSV-2) and +0.25 (VZV)). This confirms the feasibility of using the RT-PCR thermocycling program to detect and quantify the genome of RNA and DNA viruses in a single run.

COVID-19 Geographical Maps and Clinical Data Warehouse PREDIMED.

PREDIMED, Clinical Data Warehouse of Grenoble Alps University Hospital, is currently participating in daily COVID-19 epidemic follow-up via spatial and chronological analysis of geographical maps. This monitoring is aimed for cluster detection and vulnerable population discovery. Our real-time geographical representations allow us to track the epidemic both inside and outside the hospital.

Evaluation of six commercial SARS-CoV-2 rapid antigen tests in nasopharyngeal swabs: Better knowledge for better patient management?

Robust antigen point-of-care SARS-CoV-2 tests have been proposed as an efficient tool to address the COVID-19 pandemic. This requirement was raised after acknowledging the constraints that are brought by molecular biology. However, worldwide markets have been flooded with cheap and potentially underperforming lateral flow assays. Herein we retrospectively compared the overall performance of five qualitative rapid antigen SARS-CoV-2 assays and one quantitative automated test on 239 clinical swabs. While the overall sensitivity and specificity are relatively similar for all tests, concordance with molecular based methods varies, ranging from 75,7% to 83,3% among evaluated tests. Sensitivity is greatly improved when considering patients with higher viral excretion (Ct≤33), proving that antigen tests accurately distinguish infectious patients from viral shedding. These results should be taken into consideration by clinicians involved in patient triage and management, as well as by national authorities in public health strategies and for mass campaign approaches.

New steps in infliximab therapeutic drug monitoring in patients with inflammatory bowel diseases.

AIMS: Therapeutic drug monitoring (TDM) of infliximab (IFX) appears to be beneficial for patients with inflammatory bowel disease (IBD). However, the recommended target concentrations depend partly on the method used to quantify IFX. Since we recently developed a liquid chromatography-tandem mass spectrometry method to quantify IFX, we aimed to determine IFX trough concentrations (Cmin) associated with biological remission. METHODS: We retrospectively measured IFX Cmin in sera from 55 patients with IBD, on IFX maintenance therapy, and for whom demographic, biological and clinical data were collected from medical records. A threshold of IFX Cmin associated with biological remission (defined by C-reactive protein < 5 mg l-1 and faecal calprotectin <150 µg g-1 ) was determined using receiver operating characteristics analysis. RESULTS: IFX Cmin ranged from <1 mg l-1 to 57.2 mg l-1 . IFX Cmin were higher (P = 0.038) in patients with biological remission and a cut-off of IFX Cmin set to 6.2 mg l-1 was associated with biological remission (sensitivity = 0.75, 95% confidence interval 0.58-0.75; specificity = 0.61, 95% confidence interval 0.39-0.83). CONCLUSION: Liquid chromatography-tandem mass spectrometry measurement of IFX Cmin and the determination of a new threshold of IFX Cmin associated with biological remission are new steps towards IFX treatment personalization in patients with IBD.

Simultaneous Quantification of Adalimumab and Infliximab in Human Plasma by Liquid Chromatography-Tandem Mass Spectrometry.

BACKGROUND: Adalimumab (ADA) and infliximab (IFX) are therapeutic monoclonal antibodies targeting tumor necrosis factor-alpha (TNFα). They are used to treat inflammatory diseases. Clinical trials have suggested that therapeutic drug monitoring for ADA or IFX could improve treatment response and cost effectiveness. However, ADA and IFX were quantified by ELISA in all these studies, and the discrepancies between the results obtained raise questions about their reliability. We describe here the validation of a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the simultaneous quantification of ADA and IFX in human samples. METHODS: Full-length antibodies labeled with stable isotopes were added to plasma samples as an internal standard. Samples were then prepared using Mass Spectrometry Immunoassay followed by trypsin digestion before ADA and IFX quantification by LC-MS/MS. ADA and IFX were quantified in serum from patients treated with ADA (n = 21) or IFX (n = 22), and the concentrations obtained were compared with those obtained with a commercial ELISA kit. RESULTS: The chromatography run lasted 8.6 minutes, and the quantification range was 1-26 mg/L. The method was reproducible, repeatable, and accurate. For both levels of internal quality control, for ADA and IFX, interday and intraday coefficients of variation and accuracies were all within 15%, in accordance with FDA recommendations. No significant cross-contamination effect was noted. Good agreement was found between LC-MS/MS and ELISA results, for both ADA and IFX. CONCLUSIONS: This LC-MS/MS method can be used for the quantification of ADA and IFX in a single analytical run and for the optimization of LC-MS/MS resource use in clinical pharmacology laboratories.

Evaluation of the cobas® GT hepatitis C virus genotyping assay in G1-6 viruses including low viral loads and LiPA failures.

Direct-acting antiviral (DAA) drug performances depend on the viral genotype. So international recommendations give typing of the virus a prerequisite for treatment choice and patient management. Commercially available HCV genotyping kits are scarce and this analysis is often in-house using tedious PCRs and Sanger sequencing, leading to a lack of standardization. A newly commercialized HCV genotyping assay based on real-time PCR has been developed by Roche Diagnostics (Mannheim, Germany). We compared this new assay with our in-house PCRs -sequencing technique on 101 regular samples and 81 LiPA failures or low viral load samples. No genotype or 1a/1b subtype mismatch was observed. Two samples were misidentified at the subtype level without clinical impact. Three genotype 1b and two genotype 1a samples with low viral load could not be subtyped. Nevertheless, 13 (13%) samples from the regular panel and 35 (43%) from the more difficult-to-type panels failed to give results on first pass with the Roche kit. Failures were mostly associated with genotype 3 subtype a, with genotype 4 subtype non-a, or with viral loads <200 IU/mL (p = 0.0061). The workflow allowed a non-specialized technician to obtain results in less than 4 hours whereas 2 to 3 days and experienced staff were required with the in-house assay. In conclusion, the Roche cobas® HCV GT kit is easy and rapid to use and provides reliable results. The high rate of uninterpretable results particularly for low viral load samples and less frequent genotypes, and the absence of subtyping for non-genotype 1 could require sending complex samples to a specialized laboratory.

Mapping of potent and specific binding motifs, GLOGEN and GVOGEA, for integrin α1ß1 using collagen toolkits II and III.

Integrins are well characterized cell surface receptors for extracellular matrix proteins. Mapping integrin-binding sites within the fibrillar collagens identified GFOGER as a high affinity site recognized by α2ß1, but with lower affinity for α1ß1. Here, to identify specific ligands for α1ß1, we examined binding of the recombinant human α1 I domain, the rat pheochromocytoma cell line (PC12), and the rat glioma Rugli cell line to our collagen Toolkit II and III peptides using solid-phase and real-time label-free adhesion assays. We observed Mg(2+)-dependent binding of the α1 I domain to the peptides in the following rank order: III-7 (GLOGEN), II-28 (GFOGER), II-7 and II-8 (GLOGER), II-18 (GAOGER), III-4 (GROGER). PC12 cells showed a similar profile. Using antibody blockade, we confirmed that binding of PC12 cells to peptide III-7 was mediated by integrin α1ß1. We also identified a new α1ß1-binding activity within peptide II-27. The sequence GVOGEA bound weakly to PC12 cells and strongly to activated Rugli cells or to an activated α1 I domain, but not to the α2 I domain or to C2C12 cells expressing α2ß1 or α11ß1. Thus, GVOGEA is specific for α1ß1. Although recognized by both α2ß1 and α11ß1, GLOGEN is a better ligand for α1ß1 compared with GFOGER. Finally, using biosensor assays, we show that although GLOGEN is able to compete for the α1 I domain from collagen IV (IC(50) ∼3 µm), GFOGER is much less potent (IC(50) ∼90 µm), as shown previously. These data confirm the selectivity of GFOGER for α2ß1 and establish GLOGEN as a high affinity site for α1ß1.