Aurélie Truffot

Type-specific serologic detection of IgG antibodies to herpes simplex virus (HSV)-1 and HSV-2 provides important diagnostic and epidemiological information. Novel assays are under development and require rigorous evaluation before routine clinical implementation. We compared the VIDAS HSV 1&2 IgG prototype assay (bioMérieux) with the widely used LIAISON HSV-1 and HSV-2 IgG assays (DiaSorin). Western blot (WB) analysis was used as the reference method to resolve discrepant results. A total of 501 serum samples from 348 patients were analyzed, including 259 patients with PCR-confirmed HSV-1 or HSV-2 infection. WB was performed on the 89 discordant samples between the two assays. For HSV-1 IgG detection, the VIDAS prototype assay showed a sensitivity of 95.4% and a specificity of 98.6%, compared with 98.9% and 94.9%, respectively, for the LIAISON HSV-1 IgG assay. For HSV-2 IgG, the VIDAS assay demonstrated a sensitivity of 100% but a lower specificity of 90.7%, whereas the LIAISON HSV-2 IgG assay showed a lower sensitivity of 88.2% and a higher specificity of 99.4%. Approximately 30% of the samples with high HSV-1 IgG index values yielded false-positive HSV-2 IgG results with the VIDAS assay. The receiver operating characteristic curve analysis indicated that increasing the VIDAS HSV-2 positivity threshold from 0.063 to 0.137 improved specificity to 98.9% while maintaining a sensitivity of 95.4%. For HSV-1 IgG detection, the VIDAS prototype assay showed performance comparable to that of the LIAISON assay. For HSV-2 IgG, optimization of the positivity threshold may substantially improve specificity, an important consideration in low-prevalence populations. IMPORTANCE: Type-specific serology is an important complement to molecular diagnosis of herpes simplex virus (HSV) infections, helping distinguish primary, recurrent, and non-primary infections; assess neonatal transmission risk; and support patient counseling. Because western blot, the reference method, is not routinely available, the clinical value of HSV serology relies on the performance of automated immunoassays. We evaluated a novel VIDAS HSV 1&2 IgG biplex prototype assay and compared it with a widely used commercial assay. The VIDAS assay showed performance comparable to the LIAISON assay for HSV-1 IgG detection while offering simultaneous detection of HSV-1 and HSV-2 antibodies in a single test. For HSV-2 IgG, however, false-positive results were observed, particularly in samples with high HSV-1 antibody levels. Importantly, optimizing the HSV-2 positivity threshold improved specificity while maintaining high sensitivity. These findings provide data for assay optimization before commercialization and highlight the need to balance sensitivity and specificity for accurate clinical interpretation.

This study compared the analytical performance of three commercial real-time PCR assays for the detection of monkeypox virus (MPV) DNA, on their own platforms and/or on open platforms, were evaluated at the virology laboratory of Grenoble-Alpes University Hospital (CHUGA): the VIASURE monkeypox virus real-time PCR Reagents on the BD MAX System; the MPXV AMP Kit on the Alinity m system; and the Altona RealStar Zoonotic Orthopoxvirus PCR Kit 1.0 on the Argene System (EMAG, ESTREAM [Biomérieux], and LC480 [Roche]). Results obtained on these platforms were compared to those from the French National Reference Center (NRC) for orthopoxviruses. A total of 52 samples were selected by the NRC and the CHUGA: 8 dilutions of MPV culture supernatant, 2 supernatants from cowpox and vaccinia virus cultures, 32 clinical samples (20 positive/12 negative), and 10 external quality control samples from the “Quality Control for Molecular Diagnostics” (QCMD) program. The sensitivity and specificity of the three assays, compared to the NRC assay, were 100% on the samples tested. All positive samples were correctly identified as positive with all three assays, and all negative samples were negative for MPV detection. Overall, the cycle threshold (Ct) values obtained with the VIASURE, Abbott, and Altona assays were lower than those obtained with the NRC PCR in 84.38%, 81.25%, and 75.86% of positive samples, respectively. In conclusion, all three assays demonstrated good analytical performance and were suitable for MPV detection. IMPORTANCE: Mpox is an emerging zoonotic disease caused by the monkeypox virus, mostly reported in Africa. In 2022, an outbreak characterized mainly by anogenital rash in MSM (men having sex with men) occurred in Europe. In 2024, many cases of infection were reported, presenting with generalized skin rashes. Detection of monkeypox virus DNA in biological samples is crucial for diagnosis, interruption of transmission chains, treatment initiation, and vaccination. During the European outbreak in 2022, numerous companies rapidly developed qPCR kits. In France, the performance of these kits has been evaluated by the French National Reference Center (NRC) for orthopoxviruses. The Grenoble-Alpes University Hospital collaborated with the NRC to evaluate three available assays on its own platforms and/or on open platforms available in its virology laboratory. All three assays demonstrated good analytical performance and can be used with confidence.

Après une greffe rénale, l’administration d’immunosuppresseurs pour prévenir les rejets de greffe, tout en évitant un risque d’infections opportunistes, est un challenge constant. Des outils biologiques permettent de guider ces adaptations, avec sur le plan infectieux l’évaluation de l’immunité humorale (sérologies) et le suivi des charges virales (cytomégalovirus (CMV), polyomavirus BK (BKV) et Epstein-Barr virus (EBV)). Toutefois, ces outils restent insuffisants pour prédire le risque infectieux. L’apparition de nouveaux immunosuppresseurs, comme le belatacept (BELA), complexifie encore cette adaptation du traitement.L’objectif de cette thèse était d’évaluer la place des nouveaux biomarqueurs virologiques, immunologiques et pharmacologiques pour prédire les infections virales opportunistes chez les transplantés rénaux, en particulier ceux dont le traitement par le tacrolimus a été modifié pour le BELA.Le premier volet de ce travail est une revue de la littérature sur les outils et stratégies pour mieux gérer le risque infectieux et améliorer la prise en charge des patients. En particulier, les nouveaux tests d’immunomonitoring comme la charge virale du Torque Teno Virus (CV TTV) et les tests fonctionnels basés sur la sécrétion d’interféron gamma (IGRA) y sont discutés. Malgré leur inclusion dans certaines recommandations, des études supplémentaires sont nécessaires pour leur standardisation.Le deuxième volet présente le travail expérimental.La première partie porte sur la place des tests d’immunomonitoring chez les patients traités par BELA, population pour laquelle aucune étude n’a été réalisée en ce sens. Ce travail a été complété par l’évaluation de l’exposition au BELA via l’étude de sa pharmacocinétique (quantification par spectrométrie de masse). La CV TTV mesurée chez 68 patients n’a pas varié après modification du traitement. Cela suggère que les valeurs prédictives rapportées dans la littérature (avec un traitement par tacrolimus) restent valables sous BELA. De la même façon, bien qu’une baisse de l’immunité cellulaire globale (évaluée par ELISpot anti-CD3) ait été observée après passage au BELA (résultats de 112 patients), ni les réponses cellulaires anti-CMV, anti-EBV et anti-BKV, ni la CV TTV n’ont subi de modifications significatives. Aucune association entre la CV TTV ou les tests ELISpot-T IFN-y spécifiques et les rejets ou les infections opportunistes n’a été observée. En raison d’un faible nombre de réactivations virales, la prédictivité de ces tests a été difficile à évaluer. Enfin, la CV TTV dans le sang total était légèrement supérieure à celle dans le plasma, pouvant être expliqué par une localisation intra-leucocytaire du TTV.La seconde partie porte plus spécifiquement sur les tests ELISpot-T IFN-y. Premièrement, nous nous sommes demandés si l’activation des cellules T évaluée in vitro, était modifiée en présence d’immunosuppresseurs à des concentrations similaires à celles retrouvées dans le sang. Nous avons montré que la concentration intra-PBMC en tacrolimus inhibant 50 % de la réponse cellulaire évaluée in vitro par ELISpot T IFN-y était de l’ordre de 163 pg/10^6 PBMC, correspondant aux concentrations habituellement retrouvées in vivo chez les patients transplantés rénaux. Deuxièmement, nous avons comparé les réponses immunitaires antivirales obtenues avec le test IGRA-VIDAS® (BioMérieux, Lyon) et notre test ELISpot-T IFN-y. Les réponses anti-CMV et anti-EBV étaient bien corrélées. Pour la réponse anti-BKV, la réponse cellulaire évaluée par ELISpot est plus faible que pour les autres virus et la sensibilité du test IGRA-VIDAS® semble insuffisante.En conclusion, ce travail a permis de faire un état des lieux des nouvelles stratégies d’immunomonitoring chez les patients greffés rénaux, notamment pour prédire les risques de réactivations virales. Il apporte une première réponse sur la faisabilité et la place de ces tests chez les patients traités par BELA.

Monitoring cellular immune responses (CMIs) appears to be a new biomarker in managing immunocompromised patients, especially kidney transplant recipients. This study compared the performance of two interferon-gamma (IFN-γ)-based assays—enzyme-linked immunosorbent spot assay (ELISpot) and VIDAS-IGRA—for detecting T-cell responses against cytomegalovirus (CMV), Epstein-Barr virus (EBV), and polyomavirus BK (BKV). Twenty-eight blood samples from kidney transplant recipients were analyzed using both assays. CMV-specific CMI was assessed using overlapping peptide pools from pp65 and IE-1 proteins (JPT Peptide Technologies GmbH) and a CMV lysate (bioMérieux). All patients were monitored for opportunistic viral replication through qPCR for up to 6 months after blood sampling. For CMV-CMI, the agreement between both assays was 91.7%, 100%, and 80% after stimulation by pp65 peptide pool, pp65 + IE-1 peptide pools, and CMV lysate, respectively. Positive responses were consistent with CMV serology, especially for the VIDAS-IGRA assay and after activation by JPT’s PepMix. For EBV-specific responses, positive assay agreement was 92.3%, whereas the positive agreement with BKV was 80.7%. For EBV and BKV, responses were generally weaker than those for CMV (positive response if >10 spots/250,000 PBMC for EBV and >10 spots/350,000 PBMC for BKV in ELISpot or level of IFN-γ >0.08 IU/mL in VIDAS). This study highlights the importance of standardizing interferon-gamma release assays (IGRA). While VIDAS-IGRA RUO offers an open, automated, and standardized solution, responses were in agreement with ELISpot-T assay and even more when using JPT’s PepMIX. However, the cellular response to BKV is weaker, and no link with viral replication has been found. This raises the question of the value of these IGRA assays in assessing the cellular response to BKV and the need to optimize these tests. A longitudinal study to observe the evolution of the cellular response could be interesting. IMPORTANCE: This original article, a proof of concept, is the first study comparing a new interferon-gamma release assay, performed on whole blood and based on an enzyme-linked immunofluorescent assay detection technique, with a homemade enzyme-linked immunosorbent spot assay using activation by the same peptides. These results were also analyzed in relation to serology results. The inclusion of these tests in clinical guidelines for monitoring cytomegalovirus, Epstein-Barr virus, and polyomavirus BK infections requires studies to evaluate their robustness and compare them.

We report 2 cases of donor-derived West Nile virus infection in kidney transplant recipients in France. Both recipients had mild disease develop and recovered without sequelae. A more proactive screening strategy in France, particularly during periods of highest risk for West Nile virus circulation, would help reduce risk for donor-derived infections.

Cytomegalovirus (CMV) infections occur in 10%–40% of organ or stem cell transplant patients. Despite the low prevalence, CMV antiviral resistance has an important impact on patient outcomes. Guidelines for transplant recipients recommend that resistance should be suspected in cases of unchanged or increasing CMV viral loads after a minimum of 2 weeks of antiviral therapy at an appropriate dose or >6 weeks of ganciclovir exposure. Next-generation amplicon sequencing (NGS) makes it possible to directly target the genes involved in this resistance. Currently, six drugs are available, and six CMV genes (UL54-UL97-UL89-UL56-UL51-UL27 genes) can harbor mutations affecting drug efficacy. Here, we developed different primers targeting these six genes with long-range polymerase chain reaction (PCR). Based on clinical requirements, all genes or a subset could be sequenced in a single run using Oxford Nanopore technology and combined with an automatic bioinformatics pipeline to detect and report mutations. We utilized 46 blood samples, five external quality controls, and 10 mixes of two bacmids provided by the national reference center (CNR) Herpesvirus Limoges, each carrying distinct mutations. Assay performance (sensitivity, specificity, and accuracy) was evaluated through an interlaboratory exchange with CNR Herpesvirus. Long-range PCR combined with next-generation sequencing analysis enables earlier and more comprehensive discrimination of the double population and determines whether the detected single-nucleotide polymorphisms are present on single or multiple CMV strains. We developed a next-generation sequencing assay combined with eight long-range PCRs to sequence all genes involved in CMV antiviral resistance and to detect early low-frequency mutations.

SOT patients require immunosuppressors to avoid graft rejection. Therapeutic drug monitoring is insufficient to find the optimal balance with immunosuppression. The evaluation of cell-mediated immunity by enzyme-linked immunospot (ELISpot) assay enumerating interferon-gamma (IFN-γ) is increasingly use. ELISpot assays are performed on peripheral blood mononuclear cells (PBMC) isolated from blood and brought into contact with specific peptides in an immunosuppressor-free environment. This study aims to determine the in vitro diffusion of tacrolimus in PBMC and to assess whether prior in vitro incubation of PBMC with tacrolimus modifies the IFN-γ ELISpot results when assessing the T-cell immune response. PBMC from healthy volunteers were obtained. Tacrolimus was added to the ELISpot wells at increasing concentration and quantification was obtained using liquid chromatography mass spectrometry. Results showed that the in vitro PBMC diffusion rate of tacrolimus was measured at 32%. A decrease in T-cell reactivity occurred with increasing tacrolimus concentration. The intra-PBMC concentration of tacrolimus able to inhibit 50% of T-cell reactivity was 163 pg/10 ⁶ PBMC, which is in the range of the in vivo intra-PBMC concentration in SOT recipients. T-cell functional assessment using ELISpot in patients treated with immunosuppressors may require the addition of immunosuppressors in vitro to better reflect the in vivo situation.

Background: Neutralizing antibodies targeting the SARS-CoV-2 Spike protein reduce COVID-19-related risk of hospitalization, particularly in high-risk individuals. The COCOPREV-R study aimed to evaluate and compare clinical outcomes in high-risk SARS-CoV-2 patients treated with dual monoclonal antibody therapies and to identify associated virological factors. Methods: The COCOPREV-R study retrospectively collected real-world data from high-risk patients receiving Bamlanivimab/Etesevimab or Casirivimab/Imdevimab dual monoclonal antibody therapies (22 February 2021 to 15 June 2021). Results: The study included 1004 patients with COVID-19, of whom 691 received Bamlanivimab/Etesevimab and 313 received Casirivimab/Imdevimab. The alpha variant represented 90.1% of those for whom data were available. The risk of hospitalization within 30 days was lower with Bamlanivimab/Etesevimab (12.7%, CI 95% [9.9–16.3%]) compared to Casirivimab/Imdevimab (28.4%, CI 95% [22.7–35.1%) (p < 0.001). The 30-day mortality rates were comparable between both groups (p = 0.982). Analysis of SARS-CoV-2 PCR negativity showed no difference between the two treatment groups (95.2% [93.0–96.9%] and 93.5% [89.1–96.6%] until day 30, p = 0.851 for Bamlanivimab/Etesevimab and Casirivimab/Imdevimab, respectively). Among persistently positive samples with available sequencing results (n = 43), Spike protein changes occurred only in Bamlanivimab/Etesevimab (42.9%) vs. Casirivimab/Imdevimab (0.0%) groups. Q493R (25.0%) and E484K (12.5%) were the most common mutations selected by Bamlanivimab/Etesevimab in follow-up samples. Other factors (immunodepression, comorbidities, and age) did not appear to be associated with the occurrence of Spike protein mutations. Conclusions: A higher rate of hospitalization was seen with Casirivimab/Imdevimab (RONAPREVE^®) in comparison with Bamlanivimab/Etesevimab treatment, but with the emergence of Spike mutations only in the Bamlanivimab/Etesevimab group.