Temporal dynamics of RSV shedding and genetic diversity in adults during the COVID-19 pandemic in a French hospital, early 2021.

Human respiratory syncytial virus (RSV) is responsible of lower respiratory tract infections which may be severe in infants, elderly and immunocompromised adults. Europe and North-American countries have observed a massive reduction of RSV incidence during the 2020-2021 winter season. Using a systematic RSV detection coupled to SARS-CoV-2 for all adult patients admitted at the Foch hospital (Suresnes, France) between January and March 2021 (n = 11,324), only eight RSV infections in patients with prolonged RNA shedding were diagnosed. RSV whole-genome sequencing revealed that six and two patients were infected by RSV groups A and B, respectively. RSV carriage lasted from 7 to at least 30 days disregarding of RSV lineage. The most prolonged RSV shedding was observed in an asymptomatic patient. We detected novel patient-specific non-synonymous mutations in the G glycoprotein gene, including a double identical mutation in the repeated region for one patient. No additional mutation occurred in the RSV genome over the course of infection in the four patients tested for. In conclusion, our results suggest that the temporal shift in the RSV epidemic is not likely to be explained by the emergence of a high frequency, unreported variant. Moreover, prolonged RSV carriages in asymptomatic patients could play a role in virus spread.

Mutations in the 3'-PPT Lead to HIV-1 Replication without Integration.

Integration of the reverse-transcribed genome is a critical step of the retroviral life cycle. Strand-transfer inhibitors (INSTIs) used for antiretroviral therapy inhibit integration but can lead to resistance mutations in the integrase gene, the enzyme involved in this reaction. A significant proportion of INSTI treatment failures, particularly those with second-generation INSTIs, show no mutation in the integrase gene. Here, we show that replication of a selected dolutegravir-resistant virus with mutations in the 3'-PPT (polypurine tract) was effective, although no integrated viral DNA was detected, due to the accumulation of unintegrated viral DNA present as 1-LTR circles. Our results show that mutation in the 3'-PPT leads to 1-LTR circles and not linear DNA as classically reported. In conclusion, our data provide a molecular basis to explain a new mechanism of resistance to INSTIs, without mutation of the integrase gene and highlights the importance of unintegrated viral DNA in HIV-1 replication. IMPORTANCE Our work highlights the role of HIV-1 unintegrated viral DNA in viral replication. A virus, resistant to strand-transfer inhibitors, has been selected in vitro. This virus highlights a mutation in the 3'PPT region and not in the integrase gene. This mutation modifies the reverse transcription step leading to the accumulation of 1-LTR circles and not the linear DNA. This accumulation of 1-LTR circles leads to viral replication without integration of the viral genome.

Ibalizumab shows in-vitro activity against group A and group B HIV-2 clinical isolates.

OBJECTIVE: Treatment of multidrug-resistant HIV-2 is an emerging issue, because of the rapid selection of mutations at time of virological failure and the low number of antiretrovirals active on HIV-2. The aim of this study was to determine the susceptibility of HIV-2 primary isolates to ibalizumab, a long-acting monoclonal antibody that binds to CD4 that is approved for the treatment of MDR HIV-1. METHODS: In-vitro phenotypic susceptibility of 16 HIV-2 primary isolates was measured using a modified version of the ANRS peripheral blood mononuclear cells (PBMC) assay. Susceptibility to ibalizumab was assessed through 50% inhibitory concentrations and maximum percentage inhibitions (MPI), and gp105 was sequenced to look for determinants of reduced susceptibility. RESULTS: Ibalizumab inhibited viral replication of all 16 isolates, with a median IC 50 value of 0.027 µg/ml (range = 0.001-0.506 µg/ml), and a median MPI of 93%. Although two isolates presented higher IC 50 (above 0.1 µg/ml), they did not exhibit a loss of potential N-linked glycosylation sites in V5 loop, as reported in HIV-1 strains with reduced susceptibility. However, both presented shorter V1 and V2 loops than the HIV-2 reference strain. CONCLUSION: Ibalizumab inhibits HIV-2 replication, with IC 50 and MPI in the range of those reported for HIV-1. These in vitro data support the use of ibalizumab in patients with MDR HIV-2, in combination with an optimized background regimen.

In vitro analysis of the replicative capacity and phenotypic susceptibility to integrase inhibitors of HIV-2 mutants with integrase insertions.

BACKGROUND: HIV-2 resistance to integrase strand-transfer inhibitors (INSTIs) is characterized by two main pathways: (i) mutations at codons 143, 148 and155; and (ii) amino acid insertion after integrase codon 231 (231ins). OBJECTIVES: To complete INSTI resistance data on HIV-2 by determining the viral replicative capacity and INSTI phenotypic susceptibility of integrase mutants obtained through site-directed mutagenesis. METHODS: Site-directed mutants (SDMs) were constructed and viral stocks produced. Viral replicative capacity was assessed by measuring HIV-2 viral load at days 3, 7 and 14. In vitro phenotypic susceptibility was measured using the ANRS PBMC assay. RESULTS: Viruses bearing 231ins did not present impaired replicative capacity, except the 231ins GIRGK mutant. A 231ins GK SDM was resistant to raltegravir and cabotegravir, but remained susceptible to dolutegravir and bictegravir. SDMs harbouring a 5 amino acid insertion (GYKGK or SREGK) were both resistant to all INSTIs. The SDM with T97A+N155H, with or without E92Q, was resistant to all INSTIs, except bictegravir. CONCLUSIONS: These first data on the newly described resistance pathway 231ins, using site-directed mutagenesis, showed no measurable impact on viral fitness and confirmed the decreased susceptibility to a first-generation INSTI (raltegravir) and cabotegravir. Resistance to second-generation INSTIs (dolutegravir and bictegravir) occurred for mutants with a 5 amino acid 231ins.

Efficacy of Severe Acute Respiratory Syndrome Coronavirus-2 Vaccine in Patients With Thoracic Cancer: A Prospective Study Supporting a Third Dose in Patients With Minimal Serologic Response After Two Vaccine Doses.

INTRODUCTION: Coronavirus disease 2019 resulted in a 30% mortality rate in patients with thoracic cancer. Given that patients with cancer were excluded from serum antisevere acute respiratory syndrome coronavirus-2 (SARS-CoV-2) vaccine registration trials, it is still unknown whether they would develop a protective antispike antibody response after vaccination. This prospective vaccine monitoring study primarily aimed to assess humoral responses to the SARS-CoV-2 vaccine in patients with thoracic cancer. METHODS: SARS-CoV-2-spike antibodies were measured using the Abbot Architect SARS-CoV-2 immunoglobulin G immunoassay before the first injection of BNT162b2 mRNA vaccine, at week 4, and 2 to 16 weeks after the second vaccine dose administration. The factors associated with antibody response were analyzed. RESULTS: Overall, 306 patients, with a median age of 67.0 years (interquartile range: 58-74), were vaccinated. Of these, 283 patients received two vaccine doses at 28-day intervals. After a 6.7-month median follow-up, eight patients (2.6%) contracted proven symptomatic SARS-CoV-2 infection, with rapid favorable evolution. Of the 269 serologic results available beyond day 14 after the second vaccine dose administration, 17 patients (6.3%) were still negative (<50 arbitrary units/mL, whereas 34 (11%) were less than 300 arbitrary units/mL (12.5th percentile). In multivariate analysis, only age (p < 0.01) and long-term corticosteroid treatment (p = 0.01) were significantly associated with a lack of immunization. A total of 30 patients received a third vaccine dose, with only three patients showing persistently negative serology thereafter, whereas the others exhibited clear seroconversion. CONCLUSIONS: SARS-CoV2 vaccines were found to be efficient in patients with thoracic cancer, most of them being immunized after two doses. A third shot given to 1% of patients with persistent low antibody titers resulted in an 88% immunization rate.

Humoral Response Induced by Prime-Boost Vaccination with the ChAdOx1 nCoV-19 and mRNA BNT162b2 Vaccines in a Teriflunomide-Treated Multiple Sclerosis Patient.

Patients with multiple sclerosis (MS) are treated with drugs that may impact immune responses to SARS-CoV-2 vaccination. Evaluation of "prime-boost" (heterologous) vaccination regimens including a first administration of a viral vector-based vaccine and a second one of an mRNA-based vaccine in such patients has not yet been completed. Here, we present the anti-spike protein S humoral response, including the neutralizing antibody response, in a 54-year-old MS patient who had been treated with teriflunomide for the past 2 years and who received a heterologous ChAdOx1 nCoV-19/ BNT162b2 vaccination regimen. The results showed a very strong anti-S IgG response and a good neutralizing antibody response. These results show that teriflunomide did not prevent the development of a satisfactory humoral response in this MS patient after vaccination with a ChAdOx1 nCoV-19/ BNT162b2 prime-boost protocol.

Impact of Low-Dose Methotrexate-Adalimumab Combination Therapy on the Antibody Response Induced by the mRNA-1273 SARS-CoV-2 Vaccine: Case of an Elderly Patient with Rheumatoid Arthritis.

Patients with rheumatoid arthritis (RA) are treated with drugs that may impact their immune responses to SARS-CoV-2 vaccines. We describe here the anti-Spike (anti-S) IgG and neutralizing antibody responses induced by the mRNA-1273 SARS-CoV-2 vaccine in a 78-years-old patient with RA, who received a low-dose combination therapy of methotrexate and adalimumab, shortly before vaccine administration. Both near-normal and impaired immune responses to vaccines have been reported previously in patients treated with these drugs. Our case report shows that, even at low doses, combined methotrexate-adalimumab therapy can be associated with a weak immune response to the mRNA1273 vaccine in elderly patients.

High-sensitivity SARS-CoV-2 group testing by digital PCR among symptomatic patients in hospital settings.

BACKGROUND: Worldwide demand for SARS-CoV-2 RT-PCR testing is still high as testing remains central to follow the disease spread and vaccine efficacy. Group testing has been proposed as a solution to expand testing capabilities but sensitivity concerns may limit its impact on the management of the pandemic. Digital PCR (RT-dPCR) has been shown to be highly sensitive and could help by providing larger testing capabilities without compromising sensitivity. METHODS: We implemented RT-dPCR based COVID-19 group testing on a commercially available system and assay (naica® system from Stilla Technologies) and investigated the sensitivity of the method in real life conditions of a university hospital in Paris, France, in May 2020. We tested the protocol in a direct comparison with reference RT-PCR testing on 448 samples split into groups of 8, 16 and 32 samples for RT-dPCR analysis. RESULTS: Individual RT-PCR testing identified 25/448 positive samples. Using 56 groups of 8, RT-dPCR identified 23 groups as positive, corresponding to 26 positive samples by individual PCR (positive percentage agreement 95.2% [95% confidence interval: 76.2-99.9%]) and including 2 samples not detected by individual RT-PCR but confirmed positive by further investigation. 15 of 28 groups of 16 tested positive, corresponding to 25 positive samples by individual PCR (positive percentage agreement 87.5% [95% confidence interval: 61.7-98.4%]). 14 groups of 32 were fully concordant with individual PCR testing but will need to be confirmed on larger datasets. CONCLUSIONS: Our proposed approach of group testing by digital PCR has similar diagnostic sensitivity compared to individual RT-PCR testing for group up to 16 samples. This approach reduces the quantity of reagent needed by up to 80% while reducing costs and increasing capabilities of testing up to 10-fold.

Evaluation of three extraction-free SARS-CoV-2 RT-PCR assays: A feasible alternative approach with low technical requirements.

The worldwide demand for SARS-CoV-2 RT-PCR testing resulted in a shortage of diagnostic kits. RNA extraction step constitutes a major bottleneck to perform diagnostic. The aim of this study was to assess performances of different extraction-free SARS-CoV-2 RT-PCR assays compared to a reference RT-PCR assay. The panel of evaluation consisted of 94 samples: 69 positive and 25 negative for SARS-CoV-2 by reference RT-PCR. Three extraction-free RT-PCR assays were assessed: (i) PrimeDirect® Probe RT-qPCR Mix (Takara), (ii) PrimeScript®RT-PCR (Takara), and (iii) SARS-CoV-2 SANSURE®BIOTECH Novel Coronavirus (Sansure). The overall sensitivity of PrimeDirect, PrimeScript and Sansure assays was 55.1 %, 69.6 % and 69.6 %, respectively. The sensitivity increased among samples with Ct<30: 91.9 % (n = 34/37), 89.2 % (n = 33/37) and 94.6 % (n = 35/37) for PrimeDirect, PrimeScript and Sansure assays, respectively. The specificity was 88 %, 100 % and 100 % for PrimeDirect, PrimeScript and Sansure assays, respectively. In the present study, we showed a good sensitivity of extraction-free PCR assays, especially for high viral loads (Ct<30), except PrimeDirect that displayed imperfect sensitivity and specificity. Despite a lower sensitivity for low viral loads, extraction-free reagents can provide a valuable option, cheaper, easier and less reagent consuming for SARS-CoV-2 diagnostic, especially in laboratory with lower experience and equipment for molecular assays.

Earlier In Vitro Viral Production With SARS-CoV-2 Alpha Than With Beta, Gamma, B, or A.27 Variants.

Since its emergence in China at the end of 2019, SARS-CoV-2 has rapidly spread across the world to become a global public health emergency. Since then, the pandemic has evolved with the large worldwide emergence of new variants, such as the Alpha (B.1.1.7 variant), Beta (B.1.351 variant), and Gamma (P.1 variant), and some other under investigation such as the A.27 in France. Many studies are focusing on antibody neutralisation changes according to the spike mutations, but to date, little is known regarding their respective replication capacities. In this work, we demonstrate that the Alpha variant provides an earlier replication in vitro, on Vero E6 and A549 cells, than Beta, Gamma, A.27, and historical lineages. This earlier replication was associated with higher infectious titres in cell-culture supernatants, in line with the higher viral loads observed among Alpha-infected patients. Interestingly, Beta and Gamma variants presented similar kinetic and viral load than the other non-Alpha-tested variants.

No association between human herpesvirus or herpesvirus saimiri and idiopathic pulmonary fibrosis.

There is a high prevalence of human herpesviruses in lung samples of IPF patients but this does not differ from controls, neither regarding prevalence, viral load levels nor co-infection rates. Herpesvirus saimiri DNA is not detected in any lung samples. https://bit.ly/2ZrKiDJ.

Evaluation of the RealStar® SARS-CoV-2 RT-PCR kit RUO performances and limit of detection.

BACKGROUND: The use of efficient, reliable and sensitive PCR assays is a cornerstone in the race to contain the SARS-CoV-2 pandemic. In this work we performed an independent evaluation of the RealStar® SARS-CoV-2 RT-PCR Kit Researh Use Only (Altona) for SARS-CoV-2 detection. METHODS: A comparative limit of detection (LoD) assessment was performed between RealStar® SARS-CoV-2 and the currently WHO recommended RT-PCR (WHO-PCR) workflow using a quantified clinical sample. Assessment of the RealStar® SARS-CoV-2 assay was also performed using 83 primary clinical samples in comparison with the WHO-PCR. RESULTS: The RealStar® SARS-CoV-2 demonstrated a slightly higher sensitivity than the WHO recommended assay with a limit of detection at 625 copies/mL instead of 1250 copies/mL for the WHO-PCR in our conditions. The overall percent agreement between RealStar® SARS-CoV-2 and WHO-PCR on 83 clinical samples was 97.6 % (81/83) with a sensitivity at 97.8 % (45/46) and specificity at 97.3 % (36/37). No cross reaction was encountered for the other human coronaviruses (HKU1, OC43, NL63, 229E). CONCLUSIONS: In this comparison of the RealStar® SARS-CoV-2 assay with the reference WHO assay, we observed a slightly better sensitivity of the RealStar® assay. It provides a robust option for all molecular biology laboratories, with a strong real-life LoD and is compatible with various real-time PCR platforms.

Evaluation of the QIAstat-Dx Respiratory SARS-CoV-2 Panel, the First Rapid Multiplex PCR Commercial Assay for SARS-CoV-2 Detection.

In the race to contain severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), efficient detection and triage of infected patients must rely on rapid and reliable testing. In this work, we performed the first evaluation of the QIAstat-Dx respiratory SARS-CoV-2 panel (QIAstat-SARS) for SARS-CoV-2 detection. This assay is the first rapid multiplex PCR (mPCR) assay, including SARS-CoV-2 detection, and is fully compatible with a non-PCR-trained laboratory or point-of-care (PoC) testing. This evaluation was performed using 69 primary clinical samples (66 nasopharyngeal swabs [NPS], 1 bronchoalveolar lavage fluid sample [BAL], 1 tracheal aspirate sample, and 1 bronchial aspirate sample) comparing SARS-CoV-2 detection with the currently WHO-recommended reverse transcription-PCR (RT-PCR) (WHO-RT-PCR) workflow. Additionally, a comparative limit of detection (LoD) assessment was performed for QIAstat-SARS and WHO-RT-PCR using a quantified clinical sample. Compatibility of sample pretreatment for viral neutralization or viscous samples with the QIAstat-SARS system were also tested. The QIAstat-Dx respiratory SARS-CoV-2 panel demonstrated a sensitivity comparable to that of the WHO-recommended assay with a limit of detection at 1,000 copies/ml. The overall percent agreement between QIAstat-Dx SARS and WHO-RT-PCR on 69 clinical samples was 97% with a sensitivity of 100% (40/40) and specificity at 93% (27/29). No cross-reaction was encountered for any other respiratory viruses or bacteria included in the panel. The QIAstat-SARS rapid multiplex PCR panel provides a highly sensitive, robust, and accurate assay for rapid detection of SARS-CoV-2. This assay allows rapid decisions even in non-PCR-trained laboratory or point-of-care testing, allowing innovative organization.

Pharmacovirological analyses of blood and male genital compartment in patients receiving dolutegravir + lamivudine dual therapy as a switch strategy (ANRS 167 LAMIDOL trial).

OBJECTIVES: To describe plasma residual HIV viraemia, cellular HIV reservoir size, blood plasma drug concentrations and their male genital tract penetration during the maintenance dual therapy dolutegravir + lamivudine. PATIENTS AND METHODS: ANRS167 LAMIDOL enrolled 104 virologically suppressed patients to switch to dolutegravir + lamivudine. In this pharmacovirological substudy, ultrasensitive plasma viral load (USpVL) and plasma drug concentrations were measured at Day 0 (D0), Week 24 (W24) and W48 of dolutegravir + lamivudine, and HIV-DNA was measured at W-8 and W48. Semen samples were collected at D0 and W24 from 18 participants. Total and unbound blood and seminal plasma drug concentrations were measured using UPLC-MS/MS. RESULTS: Median HIV-DNA was 2.5 log10 copies/106 PBMC (IQR = 2.2-3.0, n = 100) at W-8 and 2.4 log10 copies/106 PBMC (IQR = 2.1-2.9, n = 100) at W48 (P = 0.17). The proportion of patients with undetected USpVL was 38% (n = 98), 43% (n = 98) and 49% (n = 97) at D0, W24 and W48, respectively (P = 0.08). Total and unbound plasma dolutegravir concentrations were stable between timepoints (P = 0.13) and all total plasma dolutegravir concentrations except one were adequate. Median free fraction of dolutegravir in plasma was 0.21%. Median blood plasma and seminal plasma concentrations of total dolutegravir at 24 h were 1812 ng/mL and 206 ng/mL, respectively. Median seminal plasma/blood plasma total concentration ratios were 11.6% and 2478% for dolutegravir and lamivudine, respectively. HIV-RNA (365 to 475 copies/mL) was detected in seminal plasma of one patient at D0 (5.9%) and of two patients at W24 (11.8%). CONCLUSIONS: These findings add further important information regarding the effectiveness of dolutegravir + lamivudine maintenance dual therapy in terms of plasma residual viraemia, cellular reservoir size and drug penetration in the male genital tract.

Limited HIV-2 reservoirs in central-memory CD4 T-cells associated to CXCR6 co-receptor expression in attenuated HIV-2 infection.

The low pathogenicity and replicative potential of HIV-2 are still poorly understood. We investigated whether HIV-2 reservoirs might follow the peculiar distribution reported in models of attenuated HIV-1/SIV infections, i.e. limited infection of central-memory CD4 T lymphocytes (TCM). Antiretroviral-naive HIV-2 infected individuals from the ANRS-CO5 (12 non-progressors, 2 progressors) were prospectively included. Peripheral blood mononuclear cells (PBMCs) were sorted into monocytes and resting CD4 T-cell subsets (naive [TN], central- [TCM], transitional- [TTM] and effector-memory [TEM]). Reactivation of HIV-2 was tested in 30-day cultures of CD8-depleted PBMCs. HIV-2 DNA was quantified by real-time PCR. Cell surface markers, co-receptors and restriction factors were analyzed by flow-cytometry and multiplex transcriptomic study. HIV-2 DNA was undetectable in monocytes from all individuals and was quantifiable in TTM from 4 individuals (median: 2.25 log10 copies/106 cells [IQR: 1.99-2.94]) but in TCM from only 1 individual (1.75 log10 copies/106 cells). HIV-2 DNA levels in PBMCs (median: 1.94 log10 copies/106 PBMC [IQR = 1.53-2.13]) positively correlated with those in TTM (r = 0.66, p = 0.01) but not TCM. HIV-2 reactivation was observed in the cells from only 3 individuals. The CCR5 co-receptor was distributed similarly in cell populations from individuals and donors. TCM had a lower expression of CXCR6 transcripts (p = 0.002) than TTM confirmed by FACS analysis, and a higher expression of TRIM5 transcripts (p = 0.004). Thus the low HIV-2 reservoirs differ from HIV-1 reservoirs by the lack of monocytic infection and a limited infection of TCM associated to a lower expression of a potential alternative HIV-2 co-receptor, CXCR6 and a higher expression of a restriction factor, TRIM5. These findings shed new light on the low pathogenicity of HIV-2 infection suggesting mechanisms close to those reported in other models of attenuated HIV/SIV infection models.

A New Mechanism of Resistance of Human Immunodeficiency Virus Type 2 to Integrase Inhibitors: A 5-Amino-Acid Insertion in the Integrase C-Terminal Domain.

BACKGROUND: Integrase strand transfer inhibitors (INSTIs) are crucial for the treatment of human immunodeficiency virus (HIV) type 2 infection, due to limited available therapeutic options. Recently, bictegravir has been approved for HIV-1, but no data are currently available for HIV-2. METHODS: We assessed the phenotypic susceptibility of 12 HIV-2 clinical isolates, obtained from 2 antiretroviral-naive and 10 antiretroviral-experienced patients, to 5 INSTIs (bictegravir, cabotegravir, dolutegravir, elvitegravir, and raltegravir) at the virological failure of an INSTI-based regimen. The 50% inhibitory concentrations (IC50s) were determined. Phenotypic inhibitory quotients were determined using trough INSTI plasma concentrations. RESULTS: Wild-type viruses were susceptible to the 5 INSTIs, with IC50s in the nanomolar range. Bictegravir had a lower IC50 than the other INSTIs on those HIV-2 isolates bearing major, resistance-associated mutations (codons 143, 148, and 155). We identified a new resistance profile-a 5-amino-acid insertion at codon 231 of the HIV-2 integrase (231INS)-in 6 patients at the virological failure of a raltegravir-based regimen. Those patients had adequate raltegravir concentrations, but harbored multiresistant viruses with low genotypic susceptibility scores (median = 1.5). This insertion rendered isolates highly resistant to raltegravir and elvitegravir, and moderately resistant to dolutegravir and cabotegravir. Regarding bictegravir, 2 isolates remained susceptible and 2 had a slight increase in IC50 (3- to 5-fold change). CONCLUSIONS: Our results confirm the potency of INSTI on HIV-2 clinical isolates with wild-type integrase. In addition, we identified a new resistance pathway, 231INS, selected in antiretroviral-experienced patients with multiresistant HIV-2 viruses. This highlights the need of close follow-up of those patients initiating an INSTI-based regimen.