Evaluation of two RT-PCR screening assays for identifying SARS-CoV-2 variants.

BACKGROUND: The recent emergence of new SARS CoV-2 variants (variants of concern, VOC) that spread rapidly and may lead to immune escape has emphasized the urgent need to monitor and control their spread. METHODS: We analyzed 2018 SARS-CoV-2 positive specimens collected between February 9 and March 22, 2021 using the Thermofisher® TaqPath™ COVID-19 CE-IVD RT-PCR kit (TaqPath) and the ID solutions® ID™ SARS-CoV-2/UK/SA Variant Triplex RT-PCR (ID triplex) assay to screen for VOCs. RESULTS: The ID triplex assay identified 62.8% of them as VOCs: 61.8% B.1.1.7 and 0.9% B.1.351/P.1. The agreement between the ID triplex results for B.1.1.7 and the TaqPath S gene target failure (SGTF)/ S gene target late detection (SGTL) profile for this variant agreed very well (k = 0.86). A low virus load was the main cause of discrepancies. Sequencing discordant results with both assays indicated that the TaqPath assay detected the B.1.1.7 lineage slightly better. Both assays suggested that the virus loads of B.1.1.7 variants were significantly higher than those of non-B.1.1.7 strains. Only 10/20 B1.351/P.1 strains detected with the ID triplex assay were confirmed by sequencing. CONCLUSIONS: We conclude that the SGTF/SGTL profiles identified using the TaqPath assay and ID triplex results are suitable for detecting the B.1.1.7 lineage. The ID triplex assay, which rapidly determines all three current VOCs simultaneously, could be a valuable tool for limiting virus spread by supporting contact-tracing and isolation.

COVID-19, Virology and Geroscience: A Perspective.

A new coronavirus, called SARS-CoV-2, was identified in Wuhan, China, in December 2019. The SARS-CoV-2 spread very rapidly, causing a global pandemic, Coronavirus Disease 2019 (COVID-19). Older adults have higher peak of viral load and, especially those with comorbidities, had higher COVID-19-related fatality rates than younger adults. In this Perspective paper, we summarize current knowledge about SARS-CoV-2 and aging, in order to understand why older people are more affected by COVID-19. We discuss about the possibility that the so-called "immunosenescence" and "inflammaging" processes, already present in a fraction of frail older adults, could allow the immune escape of SARS-CoV-2 leading to COVID-19 serious complications. Finally, we propose to use geroscience approaches to the field of COVID-19.

Reduced progression of bone erosion in cytomegalovirus seropositive rheumatoid arthritis patients.

BACKGROUND: Human cytomegalovirus (HCMV) seropositivity has been associated with higher inflammation during rheumatoid arthritis (RA). However, no data are available on the impact of HCMV seropositivity on bone erosion progression during RA. METHODS: We selected 487 individuals of ESPOIR cohort who fulfilled the 2010 ACR/EULAR criteria for RA. HCMV serology for these patients was determined using Architect CMV IgG assay. Baseline and 1-year central X-ray reading using modified Total Sharp Score (mTSS), Erosion Sharp Score, and joint space narrowing Sharp score were used to quantify structural damage progression. We performed univariate and multivariate analyses to investigate the association between HCMV status and bone erosion progression. RESULTS: We analyzed 273 HCMV seropositive (HCMV+) and 214 HCMV seronegative (HCMV-) RA patients. At inclusion, HCMV+ patients were less frequently ACPA+ (49.8% versus 58.9%, p < 0.0465) and had a higher DAS28-ESR (5.55 ± 1.24 versus 5.20 ± 1.14, p < 0.0013) in comparison with HCMV-. At 1 year, bone erosion progression (delta erosion Sharp score > 1 point) was lower in HCMV+ patients (16.1% versus 25.2%, p = 0.0128) in comparison with HCMV-. HCMV+ status remained independently associated with lower bone erosion progression in multivariate analysis. CONCLUSIONS: Our findings suggest that, independently of other confounding factors, HCMV seropositivity is associated with a lower progression of bone erosion during RA.

Increased CXCR3+ T Cells Impairs Recruitment of T-Helper Type 17 Cells via Interferon γ and Interleukin 18 in the Small Intestine Mucosa During Treated HIV-1 Infection.

The restoration of CD4+ T cells, especially T-helper type 17 (Th17) cells, remains incomplete in the gut mucosa of most human immunodeficiency virus type 1 (HIV-1)-infected individuals despite sustained antiretroviral therapy (ART). Herein, we report an increase in the absolute number of CXCR3+ T cells in the duodenal mucosa during ART. The frequencies of Th1 and CXCR3+ CD8+ T cells were increased and negatively correlated with CCL20 and CCL25 expression in the mucosa. In ex vivo analyses, we showed that interferon γ, the main cytokine produced by Th1 and effector CD8+ T cells, downregulates the expression of CCL20 and CCL25 by small intestine enterocytes, while it increases the expression of CXCL9/10/11, the ligands of CXCR3. Interleukin 18, a pro-Th1 cytokine produced by enterocytes, also contributes to the downregulation of CCL20 expression and increases interferon γ production by Th1 cells. This could perpetuate an amplification loop for CXCR3-driven Th1 and effector CD8+ T cells recruitment to the gut, while impairing Th17 cells homing through the CCR6-CCL20 axis in treated HIV-1-infected individuals.

[HEV and transfusion-recipient risk]. / Virus de l'hépatite E et risque de transmission par transfusion.

HEV infections are mainly food- and water-borne but transfusion-transmission has occurred in both developing and developed countries. The infection is usually asymptomatic but it can lead to fulminant hepatitis in patients with underlying liver disease and pregnant women living in developing countries. It also causes chronic hepatitis E, with progressive fibrosis and cirrhosis, in approximately 60 % of immunocompromised patients infected with HEV genotype 3. Extra-hepatic manifestations such as neurological and renal manifestations have been reported. The risk of a transfusion-transmitted HEV infection is linked to the frequency of viremia in blood donors, the donor virus load and the volume of plasma in the final transfused blood component. Several developed countries have adopted measures to improve blood safety based on the epidemiology of HEV.

HEV and transfusion-recipient risk.

HEV infections are mainly food- and water-borne but transfusion-transmission has occurred in both developing and developed countries. The infection is usually asymptomatic but it can lead to fulminant hepatitis in patients with underlying liver disease and pregnant women living in developing countries. It also causes chronic hepatitis E, with progressive fibrosis and cirrhosis, in approximately 60% of immunocompromised patients infected with HEV genotype 3. The risk of a transfusion-transmitted HEV infection is linked to the frequency of viremia in blood donors, the donor virus load and the volume of plasma in the final transfused blood component. Several developed countries have adopted measures to improve blood safety based on the epidemiology of HEV.

Parsonage-Turner syndrome due to autochthonous acute genotype 3f hepatitis E virus infection in a nonimmunocompromised 55-year-old patient.

Hepatitis E virus (HEV) infection is an emerging autochthonous disease in industrialized countries. Extra-hepatic manifestations, in particular neurologic manifestations, have been reported in HEV infection. Only a few cases of hepatitis E-associated Parsonage-Turner syndrome have been reported, and HEV genotypes were rarely determined. Here, we report the case of a Parsonage-Turner syndrome associated with an acute autochthonous HEV infection in a 55-year-old immunocompetent patient. HEV genomic RNA was detected in serum and cerebrospinal fluid samples (CSF), and molecular phylogenetic analysis of HEV was performed. The interest of this case lies in its detailed description notably the molecular analysis of HEV RNA isolated from serum and CSF. HEV infection should be considered in diagnostic investigations of neurologic manifestations associated with liver function perturbations.

Prevalence of HIV-1 drug resistance in treated patients with viral load >50 copies/mL: a 2014 French nationwide study.

Background: Surveillance of HIV-1 resistance in treated patients with a detectable viral load (VL) is important to monitor, in order to assess the risk of spread of resistant viruses and to determine the proportion of patients who need new antiretroviral drugs with minimal cross-resistance. Methods: The HIV-1 protease and reverse transcriptase (RT) and integrase genes were sequenced in plasma samples from 782 consecutive patients on failing antiretroviral regimens, seen in 37 specialized centres in 2014. The genotyping results were interpreted using the ANRS v24 algorithm. Prevalence rates were compared with those obtained during a similar survey conducted in 2009. Results: The protease and RT sequences were obtained in 566 patients, and the integrase sequence in 382 patients. Sequencing was successful in 60%, 78%, 78% and 87% of patients with VLs of 51-200, 201-500, 501-1000 and >1000 copies/mL, respectively. Resistance to at least one antiretroviral drug was detected in 56.3% of samples. Respectively, 3.9%, 8.7%, 1.5% and 3.4% of patients harboured viruses that were resistant to any NRTI, NNRTI, PI and integrase inhibitor (INI). Resistance rates were lower in 2014 than in 2009. Resistance was detected in 48.5% of samples from patients with a VL between 51 and 200 copies/mL. Conclusion: In France in 2014, 90.0% of patients in AIDS care centres were receiving antiretroviral drugs and 12.0% of them had VLs >50 copies/mL. Therefore, this study suggests that 6.7% of treated patients in France might transmit resistant strains. Resistance testing may be warranted in all treated patients with VL > 50 copies/mL.

Detection and quantification of HIV-1 RNA with a fully automated transcription-mediated-amplification assay.

BACKGROUND: Nucleic acid testing is the major method used to monitor HIV viral load. Commercial systems based on real-time PCR assays are available for high-volume centralized laboratory testing, but they are not fully automated. OBJECTIVES AND STUDY DESIGN: We have compared the diagnostic performance of the Hologic Aptima HIV-1 Quant Dx assay (Aptima) (based on real-time TMA) on the Panther instrument, a fully-automated random access platform, to that of, the Roche Cobas Ampliprep Cobas TaqMan (CAP/CTM) HIV-1 version 2.0 (based on real-time PCR). RESULTS: Probit analysis of replicate dilutions of NIBSC WHO International HIV-1 Standard, gave LODs of 8.6 c/ml for Aptima and 15.2 c/ml for CAP/CTM. The agreement between the assays was excellent when measuring HIV RNA in a calibrated reference (κ=0.90, p<0.001) and good when measuring clinical samples (κ=0.62, p<0.001). The correlation among the samples quantified by the two methods was very good (r=0.95, p<0.001) and the mean difference between the values obtained with the two assays was 0.02 log c/ml for B and non-B subtypes. The vast majority of results showed <0.5 log variance between the two assays (89%); only one sample showed results that differed by over 1.0 log c/ml. CONCLUSION: The performance of the new fully automated Aptima assay is adequate for clinical monitoring of HIV-1 RNA during infections and treatment. The Aptima assay is well suited for routine laboratory use.

Performance of a completely automated system for monitoring CMV DNA in plasma.

BACKGROUND: Completely automated systems for monitoring CMV-DNA in plasma samples are now available. OBJECTIVES: Evaluate analytical and clinical performances of the VERIS™/MDx System CMV Assay(®). STUDY DESIGN: Analytical performance was assessed using quantified quality controls. Clinical performance was assessed by comparison with the COBAS(®) Ampliprep™/COBAS(®) Taqman CMV test using 169 plasma samples that had tested positive with the in-house technique in whole blood. RESULTS: The specificity of the VERIS™/MDx System CMV Assay(®) was 99% [CI 95%: 97.7-100]. Intra-assay reproducibilities were 0.03, 0.04, 0.05 and 0.04 log10IU/ml (means 2.78, 3.70, 4.64 and 5.60 log10IU/ml) for expected values of 2.70, 3.70, 4.70 and 5.70 log10IU/ml. The inter-assay reproducibilities were 0.12 and 0.08 (means 6.30 and 2.85 log10IU/ml) for expected values of 6.28 and 2.80 log10IU/ml. The lower limit of detection was 14.6IU/ml, and the assay was linear from 2.34 to 5.58 log10IU/ml. The results for the positive samples were concordant (r=0.71, p<0.0001; slope of Deming regression 0.79 [CI 95%: 0.56-1.57] and y-intercept 0.79 [CI 95%: 0.63-0.95]). The VERIS™/MDx System CMV Assay(®) detected 18 more positive samples than did the COBAS(®) Ampliprep™/COBAS(®) Taqman CMV test and the mean virus load were higher (0.41 log10IU/ml). Patient monitoring on 68 samples collected from 17 immunosuppressed patients showed similar trends between the two assays. As secondary question, virus loads detected by the VERIS™/MDx System CMV Assay(®) were compared to those of the in-house procedure on whole blood. The results were similar between the two assays (-0.09 log10IU/ml) as were the patient monitoring trends. CONCLUSION: The performances of the VERIS™/MDx System CMV Assay(®) facilitated its routine use in monitoring CMV-DNA loads in plasma samples.

New insights into the natural history of hepatitis E virus infection through a longitudinal study of multitransfused immunocompetent patients in France.

Little is known about the natural history of Hepatitis E virus (HEV) infection in immunocompetent individuals. The prevalence, the course of infection and the occurrence of transmission by transfusion were investigated in multitransfused immunocompetent patients/blood donor pairs included in a longitudinal sample repository collection and followed up between 1988 and 2010. Ninety-eight subjects aged 6-89 years and suffering from acquired haemoglobinopathies were tested for HEV markers (IgM, IgG and RNA) in serial samples collected every 2 or 3 years. Eighteen patients (18.4%) were positive for HEV-IgG at baseline with a prevalence increasing from 12.5% below 26 years to 32% above 56 years. Nine patients remained IgG positive along the study and nine lost their antibodies after a mean follow-up of 7.4 years (1-22 years). One seropositive patient showed an increase of IgG level and RNA-HEV reappearance 1 year after inclusion, suggesting a reinfection and one seroconversion, probably acquired through blood transfusion was observed. This first longitudinal study including immunocompetent individuals confirms that HEV infection is common in Western Europe and that transfusion transmission occurs probably less frequently than expected. In addition, seroreversion and reinfection seem to be common. This suggests that the anti-HEV may not persist overtime naturally. However, repeat exposure to the virus related to the high prevalence of HEV infection may result in a sustainable specific IgG response.

CCR6(-) regulatory T cells blunt the restoration of gut Th17 cells along the CCR6-CCL20 axis in treated HIV-1-infected individuals.

The gut CD4(+) T cells, particularly the T helper type 17 (Th17) subset, are not completely restored in most HIV-1-infected individuals despite combined antiretroviral therapy, when initiated at the chronic phase of infection. We show here that the CCR6-CCL20 chemotactic axis is altered, with reduced CCL20 production by small intestine epithelial cells in treated HIV-1-infected individuals. This leads to impaired CCR6(+)CD4(+) T-cell homing, particularly Th17 cells, to the small intestine mucosa. In contrast, the frequency of gut FoxP3(+) T regulatory (Treg) cells, specifically the CCR6(-) subset, was increased. The resulting imbalance in the Th17/CCR6(-) Treg ratio and the associated shift from interleukin (IL)-17 to IL-10 and transforming growth factor-ß (TGF-ß) blunts CCL20 production by enterocytes, perpetuating a negative feedback for the recruitment of CCR6(+)CD4(+) T cells to the small intestine in treated HIV-1-infected individuals.

Efficacy and Safety of Sofosbuvir-Based Antiviral Therapy to Treat Hepatitis C Virus Infection After Kidney Transplantation.

There is no approved therapy for hepatitis C virus (HCV) infection after kidney transplantation, and no data regarding the use of new-generation direct antiviral agents (DAAs) have been published so far. The aims of this pilot study were to assess the efficacy and safety of an interferon-free sofosbuvir-based regimen to treat chronic HCV infection in kidney transplant recipients. Twenty-five kidney transplant recipients with chronic HCV infection were given, for 12 (n = 19) or 24 weeks (n = 6), sofosbuvir plus ribavirin (n = 3); sofosbuvir plus daclatasvir (n = 4); sofosbuvir plus simeprevir, with (n = 1) or without ribavirin (n = 6); sofosbuvir plus ledipasvir, with (n = 1) or without ribavirin (n = 9); and sofosbuvir plus pegylated-interferon plus ribavirin (n = 1). A rapid virological response, defined by undetectable viremia at week 4 after starting DAA therapy, was observed in 22 of the 25 patients (88%). At the end of therapy, HCV RNA was undetectable in all patients. At 4 and 12 weeks after completing DAA therapy, all had a sustained virological response. The tolerance to anti-HCV therapy was excellent and no adverse event was observed. A significant decrease in calcineurin inhibitor levels was observed after HCV clearance. New-generation oral DAAs are efficient and safe to treat HCV infection after kidney transplantation.

Neurologic Disorders in Immunocompetent Patients with Autochthonous Acute Hepatitis E.

Neurologic disorders, mainly Guillain-Barré syndrome and Parsonage­Turner syndrome (PTS), have been described in patients with hepatitis E virus (HEV) infection in industrialized and developing countries. We report a wider range of neurologic disorders in nonimmunocompromised patients with acute HEV infection. Data from 15 French immunocompetent patients with acute HEV infection and neurologic disorders were retrospectively recorded from January 2006 through June 2013. The disorders could be divided into 4 main entities: mononeuritis multiplex, PTS, meningoradiculitis, and acute demyelinating neuropathy. HEV infection was treated with ribavirin in 3 patients (for PTS or mononeuritis multiplex). One patient was treated with corticosteroids (for mononeuropathy multiplex), and 5 others received intravenous immunoglobulin (for PTS, meningoradiculitis, Guillain-Barré syndrome, or Miller Fisher syndrome). We conclude that pleiotropic neurologic disorders are seen in HEV-infected immunocompetent patients. Patients with acute neurologic manifestations and aminotransferase abnormalities should be screened for HEV infection.

Hepatitis E virus in patients with decompensated chronic liver disease: a prospective UK/French study.

BACKGROUND: In developed countries, hepatitis E is a porcine zoonosis caused by hepatitis E virus (HEV) genotype 3. In developing countries, hepatitis E is mainly caused by genotype 1, and causes increased mortality in patients with pre-existing chronic liver disease (CLD). AIM: To determine the role of HEV in patients with decompensated CLD. METHODS: Prospective HEV testing of 343 patients with decompensated CLD at three UK centres and Toulouse France, with follow-up for 6 months or death. IgG seroprevalence was compared with 911 controls. RESULTS: 11/343 patients (3.2%) had acute hepatitis E infection, and three died. There were no differences in mortality (27% vs. 26%, OR 1.1, 95% CI 0.28-4.1), age (P = 0.9), bilirubin (P = 0.5), alanine aminotransferase (P = 0.06) albumin (P = 0.5) or international normalised ratio (P = 0.6) in patients with and without hepatitis E infection. Five cases were polymerase chain reaction (PCR) positive (genotype 3). Hepatitis E was more common in Toulouse (7.9%) compared to the UK cohort (1.2%, P = 0.003). HEV IgG seroprevalence was higher in Toulouse (OR 17, 95% CI 9.2-30) and Truro (OR 2.5, 95% CI 1.4-4.6) than in Glasgow, but lower in cases, compared to controls (OR 0.59, 95% CI 0.41-0.86). CONCLUSIONS: Hepatitis E occurs in a minority of patients with decompensated chronic liver disease. The mortality is no different to the mortality in patients without hepatitis E infection. The diagnosis can only be established by a combination of serology and PCR, the yield and utility of which vary by geographical location.

Identification of a duplicated V3 domain in NS5A associated with cirrhosis and hepatocellular carcinoma in HCV-1b patients.

BACKGROUND: The NS5A protein of the hepatitis C virus has been shown to be involved in the development of hepatocellular carcinoma. OBJECTIVES: In a French multicenter study, we investigated the clinical and epidemiological features of a new HCV genotype 1b strain bearing a wide insertion into the V3 domain. STUDY DESIGN: We studied NS5A gene sequences in 821 French patients infected with genotype 1b HCV. RESULTS: We identified an uncharacterized V3 insertion without ORF disruption in 3.05% of the HCV sequences. The insertion comprised 31 amino-acids for the majority of patients; 3 patients had 27 amino-acids insertions and 1 had a 12 amino-acids insertion. Sequence identity between the 31 amino-acids insertions and the V3 domain ranged from 48 to 96% with E-values above 4e(-5), thus illustrating sequence homology and a partial gene duplication event that to our knowledge has never been reported in HCV. Moreover we showed the presence of the duplication at the time of infection and its persistence at least during 12 years in the entire quasispecies. No association was found with extrahepatic diseases. Conversely, patients with cirrhosis were two times more likely to have HCV with this genetic characteristic (p=0.04). Moreover, its prevalence increased with liver disease severity (from 3.0% in patients without cirrhosis to 9.4% in patients with both cirrhosis and HCC, p for trend=0.045). CONCLUSIONS: We identified a duplicated V3 domain in the HCV-1b NS5A protein for the first time. The duplication may be associated with unfavorable evolution of liver disease including a possible involvement in liver carcinogenesis.

Seroprevalence in blood donors reveals widespread, multi-source exposure to hepatitis E virus, southern France, October 2011.

The apparent seroprevalence of hepatitis E Virus (HEV)varies greatly among developed countries depending on the geographical area and the sensitivity of immunoassays. We used a validated assay to determine the prevalence of HEV IgG and IgM antibodies among 3,353 blood donors living in southern France,who gave blood during the two first weeks of October 2011 and participated in the study. Demographic and epidemiological information was collected using aspecific questionnaire. We also screened 591 samples for HEV RNA. Overall IgG seroprevalence was 39.1%and varied from 20% to 71.3% depending on the geographical area (p < 0.001) while IgM seroprevalence was 3.31%. Anti-HEV IgG was significantly correlated with increasing age (p < 0.001), eating uncooked pork liver sausages (p < 0.001), offal (p = 0.003), or mussels(p = 0.02). Anti-HEV IgM was associated with being male (p = 0.01) and eating uncooked pork liver sausages(p = 0.02). HEV RNA was detected in one of the 99 anti-HEV IgM-positive samples, but in none of the 492 anti-HEV IgM-negative samples. HEV is hyperendemic in southern France. Dietary and culinary habits alone cannot explain the epidemiology of HEV in this region, indicating that other modes of contamination should be investigated.