Prolonged replication of BA.1 and BA.2 Omicron lineages compared to Delta variant in nasopharyngeal samples from COVID-19 patients.

OBJECTIVES: We aimed to characterize and compare the viral loads (VL) of the Omicron BA.1 and BA.2 lineages and the Delta variant in nasopharyngeal samples from newly diagnosed COVID-19 patients and their kinetics over time. PATIENTS AND METHODS: The kinetics of the VL were measured on the CT data from 215 SARS-CoV-2 positive patients who presented at least two positive PCRs a day apart and were screened for SARS-CoV-2 viral lineages. RESULTS: We observed no significant difference in median CT value during the first diagnostic test between the Delta variant and the two Omicron lineages. However, the kinetics of CT decreases for the BA.1 and BA.2 lineage were significantly lengthier in time than the kinetics for the Delta variant. The BA.2 lineage presented lower median CT value (-2 CT) (inversely proportional to the VL) than the BA.1 lineage. CONCLUSIONS: BA.2 Omicron lineage presented higher VL than BA.1 Omicron lineage at diagnostic. Omicron BA.1 and BA.2 lineages have more prolonged replication than the Delta variant.

Validation of the Performance of A1HPV6, a Triage Blood Test for the Early Diagnosis and Prognosis of SARS-CoV-2 Infection.

BACKGROUND AND AIMS: Apolipoprotein A1 (A1) and haptoglobin (HP) serum levels are associated with the spread and severity of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. We have constructed and validated a multivariable risk calculator (A1HPV6) integrating A1, HP, alpha2-macroglobulin, and gamma glutamyl transferase to improve the performances of virological biomarkers. METHODS: In a prospective observational study of hospitalized patients with nonsevere SARS-CoV-2 infection, A1HPV6 was constructed in 127 patients and validated in 116. The specificity was assessed in 7482 controls representing the general population. The primary diagnostic endpoint was the area under the receiver operating characteristic curve in patients with positive SARS-CoV-2 PCR. The primary prognostic endpoint was the age-and sex-adjusted risk of A1HPV6 to predict patients with WHO-stage > 4 (W > 4) severity. We assessed the kinetics of the A1HPV6 components in a nonhuman primate model (NHP), from baseline to 7 days (D7) after SARS-CoV-2 infection. RESULTS: The area under the receiver operating characteristic curve for A1HPV6 was 0.99 (95% CI 0.97-0.99) in the validation subset, which was not significantly different from that in the construction subset, 0.99 (0.99-0.99; P = .80), like for sensitivity 92% (85-96) vs 94% (88-97; P = .29). A1HPV6 was associated with W > 4, with a significant odds ratio of 1.3 (1.1-1.5; 0.002). In NHP, A1 levels decreased (P < .01) at D2 and normalized at D4; HP levels increased at D2 and peaked at D4. In patients, A1 concentration was very low at D2 vs controls (P < .01) and increased at D14 (P < .01) but was still lower than controls; HP increased at D2 and remained elevated at D14. CONCLUSION: These results validate the diagnostic and prognostic performances of A1HPV6. Similar kinetics of apolipoprotein A1, HP, and alpha-2-macroglobulin were observed in the NHP model. ClinicalTrials.gov number, NCT01927133.

Accuracy assessment of total or IgG Immunoglobulin to hepatitis A virus tests around immunity threshold.

BACKGROUND: Anti-hepatitis A virus (HAV) antibody titers at 20 IU/L are assumed to correlate with protection against HAV challenge. METHODS: We examined the accuracy and precision of currently in use immunoassays for total or anti-HAV IgG determination, by repeated testing of dilutions of the international anti-HAV standard, within a 10-50 IU/mL concentration range. RESULTS AND CONCLUSION: Eight immunoassays were evaluated. All could confidently identify people who need to be vaccinated, or who might benefit from a booster vaccine: no positive interpretation for the 10 and 15 IU/mL concentrations. However, qualitative interpretation may differ from test to test in the 15-30 IU/mL range. This variation has to be taken into account when comparing seroprevalence data.

External validation of LCR1-LCR2, a multivariable HCC risk calculator, in patients with chronic HCV.

BACKGROUND & AIMS: The Liver Cancer Risk test algorithm (LCR1-LCR2) is a multianalyte blood test combining proteins involved in liver cell repair (apolipoprotein-A1 and haptoglobin), known hepatocellular carcinoma (HCC) risk factors (sex, age, and gamma-glutamyl transferase), a marker of fibrosis (alpha2-macroglobulin) and alpha-fetoprotein (AFP), a specific marker of HCC. The aim was to externally validate the LCR1-LCR2 in patients with chronic HCV (CHC) treated or not with antivirals. METHODS: Pre-included patients were from the Hepather cohort, a multicentre prospective study in adult patients with CHC in France. LCR1-LCR2 was assessed retrospectively in patients with the test components and AFP, available at baseline. The co-primary study outcome was the negative predictive value (NPV) of LCR1-LCR2 for the occurrence of HCC at 5 years and for survival without HCC according to the predetermined LCR1-LCR2 cut-offs. The cut-offs were adjusted for risk covariables and for the response to HCV treatment, and were quantified using time-dependent proportional hazards models. RESULTS: In total, 4,903 patients, 1,026 (21.9%) with baseline cirrhosis, were included in the study. Patients were followed for a median of 5.7 (IQR 4.2-11.3) years. A total of 3,788/4,903 (77.3%) patients had a sustained virological response. There were 137 cases of HCC at 5 years and 214 at the end of follow-up. HCC occurred at 5 years in 24/3,755 patients with low-risk LCR1-LCR2 compared with 113/1,148 patients with high-risk LCR1-LCR2. The NPV was 99.4% (95% CI 99.1-99.6). Similar findings (hazard ratio, 10.8; 95% CI, 8.1-14.3; p <0.001) were obtained after adjustment for exposure to antivirals, age, sex, geographical origin, HCV genotype 3, alcohol consumption, and type 2 diabetes mellitus. CONCLUSIONS: The results showed that LCR1-LCR2 can be used to successfully identify patients with HCV at very low risk of HCC at 5 years. LAY SUMMARY: Hepatocellular carcinoma (HCC) is the fourth leading cause of cancer-related death worldwide and the fastest growing cause of cancer death in many countries. We constructed and internally validated a new multianalyte blood test to assess this Liver Cancer Risk (LCR1-LCR2). This study confirmed the performance of LCR1-LCR2 in patients with chronic HCV in the national French cohort Hepather, and its ability to identify patients at a very low risk of HCC at 5 years. CLINICAL TRIALS REGISTRATION: The study is registered at ClinicalTrials.gov (NCT01953458).

Performance of 30 commercial SARS-CoV-2 serology assays in testing symptomatic COVID-19 patients.

We report evaluation of 30 assays' (17 rapid tests (RDTs) and 13 automated/manual ELISA/CLIA assay (IAs)) clinical performances with 2594 sera collected from symptomatic patients with positive SARS-CoV-2 rRT-PCR on a respiratory sample, and 1996 pre-epidemic serum samples expected to be negative. Only 4 RDT and 3 IAs fitted both specificity (> 98%) and sensitivity (> 90%) criteria according to French recommendations. Serology may offer valuable information during COVID-19 pandemic, but inconsistent performances observed among the 30 commercial assays evaluated, which underlines the importance of independent evaluation before clinical implementation.

Performance of serum apolipoprotein-A1 as a sentinel of Covid-19.

BACKGROUND: Since 1920, a decrease in serum cholesterol has been identified as a marker of severe pneumonia. We have assessed the performance of serum apolipoprotein-A1, the main transporter of HDL-cholesterol, to identify the early spread of coronavirus disease 2019 (Covid-19) in the general population and its diagnostic performance for the Covid-19. METHODS: We compared the daily mean serum apolipoprotein-A1 during the first 34 weeks of 2020 in a population that is routinely followed for a risk of liver fibrosis risk in the USA (212,297 serum) and in France (20,652 serum) in relation to a local increase in confirmed cases, and in comparison to the same period in 2019 (266,976 and 28,452 serum, respectively). We prospectively assessed the sensitivity of this marker in an observational study of 136 consecutive hospitalized cases and retrospectively evaluated its specificity in 7,481 controls representing the general population. RESULTS: The mean serum apolipoprotein-A1 levels in the survey populations began decreasing in January 2020, compared to the same period in 2019. This decrease was highly correlated with the daily increase in confirmed Covid-19 cases in the following 34 weeks, both in France and USA, including the June and mid-July recovery periods in France. Apolipoprotein-A1 at the 1.25 g/L cutoff had a sensitivity of 90.6% (95%CI84.2-95.1) and a specificity of 96.1% (95.7-96.6%) for the diagnosis of Covid-19. The area under the characteristics curve was 0.978 (0.957-0.988), and outperformed haptoglobin and liver function tests. The adjusted risk ratio of apolipoprotein-A1 for survival without transfer to intensive care unit was 5.61 (95%CI 1.02-31.0; P = 0.04). CONCLUSION: Apolipoprotein-A1 could be a sentinel of the pandemic in existing routine surveillance of the general population. NCT01927133, CER-2020-14.

High clustering of acute HCV infections and high rate of associated STIs among Parisian HIV-positive male patients.

BACKGROUND: Increasing incidence of hepatitis C virus (HCV) infection in human immunodeficiency virus (HIV)-positive men having sex with men (MSM) has been described in recent years. Phylogenetic analyses of acute HCV infections were undertaken to characterize the dynamics during the epidemic in Paris, and associated sexually transmitted infections (STIs) were evaluated. METHODS: Sanger sequencing of polymerase gene was performed. Maximum likelihood phylogenies were reconstructed using FastTree 2.1 under a GTR+CAT model. Transmission chains were defined as clades with a branch probability ≥0.80 and intraclade genetic distances <0.02 nucleotide substitutions per sites. STIs detected ≤1 month before HCV diagnosis were considered. RESULTS: Among the 85 studied patients, at least 81.2% were MSM. Respectively, 47.6%, 39.0%, 11.0% and 2.4% were infected with genotypes 1a, 4d, 3a and 2k. At least 91.8% were co-infected with HIV. HCV re-infection was evidenced for 24.7% of patients and STIs for 20.0% of patients. Twenty-two transmission chains were identified, including 52 acute hepatitis C (11 pairs and 11 clusters from three to seven patients). CONCLUSIONS: These results revealed strong clustering of acute HCV infections. Thus, rapid treatment of both chronic and acute infections is needed among this population to decrease the prevalence of HCV, in combination with preventive behavioural interventions.

Net emergence of substitutions at position 28 in NS5A of hepatitis C virus genotype 4 in patients failing direct-acting antivirals detected by next-generation sequencing.

More data on resistance of HCV genotype (GT) 3 and 4 to direct-acting antivirals (DAAs) are still needed. Here we investigated the presence of resistance-associated substitutions (RASs) pre- and post-treatment and their emergence under DAAs in HCV GT3- and GT4-infected patients failing DAA regimens by next-generation sequencing (NGS). Sanger sequencing and NGS were performed on NS5B and NS5A in plasma samples prior to and post treatment of 13 patients. Positions implicated in resistance to anti-NS5A and anti-NS5B in the literature were analysed. No baseline RASs was detected in NS5B but one GT4r virus developed the mutation S282T at failure. In NS5A, pre-existing RASs or polymorphisms were detected in viruses of 6/10 patients (L28M for a GT4a, M28V for a GT4r, L30R for a GT4a, 2 GT4d and 1 GT4r, and T58P for a GT4d) by Sanger sequencing and in viruses of 7/10 patients by NGS. Additional baseline minority substitutions detected by NGS were Y93H in a GT3a, L28M in a GT4a and GT4d, and L28F in a GT4d virus. At failure, these substitutions were found at a frequency of 100%. Y93H was detected alone at baseline, whilst L28M and L28F were accompanied by polymorphisms L30R or L30R + T58P. Use of NGS in patients failing DAAs and infected by HCV GT3 and GT4 revealed the emergence of specific patterns of substitutions in NS5A and NS5B, in particular substitutions at position 28 in NS5A in GT4 virus, highlighting the need to list these substitutions in guidelines for resistance interpretation.

Chronic hepatitis E in a heart transplant patient: sofosbuvir and ribavirin regimen not fully effective.

Hepatitis E virus (HEV) can induce chronic infections in the case of immunosuppression, which are sometimes not cured with ribavirin. Furthermore, sofosbuvir is a highly potent inhibitor of HCV polymerase and was shown to inhibit HEV genotype-3 replication in vitro. We report here the outcome of sofosbuvir/ribavirin therapy on a chronic HEV infection in a heart transplant recipient non-responder to ribavirin. After 24 weeks, the regimen failed to cure the persistent HEV infection, highlighting the need of therapeutic options for HEV-infected immunosuppressed patients.

Multicenter comparison of the new Cobas 6800 system with Cobas Ampliprep/Cobas TaqMan and Abbott RealTime for the quantification of HIV, HBV and HCV viral load.

BACKGROUND: The new Roche Cobas 6800 platform (C6800) has been recently introduced for viral load (VL) measurement. OBJECTIVES: Comparing C6800 to Cobas Ampliprep/Cobas TaqMan v2.0 (CAP/CTM) for the quantification of HIV, HBV and HCV viremia, and to the Abbott RealTime assay (ABB) for HCV quantification. STUDY DESIGN: We analysed 121 samples for HBV, and 139 for HIV-1 including 2 groupO and 137 group M viruses (36.5% subtype B, 27.0% CRF02_AG, 22.6% from other clades, and 14% subtype not available). For the 100 HCV samples compared with CAP/CTM, 42% were genotype 1 and 17% were genotype 4. For the 68 HCV samples compared with ABB, 52.9% were genotype 1 and 22.1% were genotype 4. RESULTS: C6800 results correlated well with those of CAP/CTM for all three viruses (R2: 0.97-0.99). However, C6800 can yield different viraemia results: higher for HIV (mean difference: +0.11 log10copies/mL, p<0.0001), and lower for HBV (mean difference:-0.11 log10 IU/mL, p<0.0001). Differences exceeded 0.5 log10 for 6.5% of HIV-1 samples and 7.4% of HBV samples. For HCV quantification, C6800 gave mostly lower values than the other assays towards the bottom of the range, and higher values in the upper part of the range, especially in comparisons with ABB, for which 28% of differences exceeded 0.5 log10 IU/mL. No particular HCV genotype was identified as responsible for these differences. CONCLUSION: Overall, the comparison tests between C6800 and CAP/CTM systems are satisfactory for the three viruses. Frequent discrepancies were observed between C6800 and ABB for HCV.

Hepatitis E infection in patients with severe alcoholic hepatitis: is there a place for systematic screening?

BACKGROUND: One study has suggested that markers of acute hepatitis E virus (HEV) infection are present in 3.6% of patients with severe alcoholic hepatitis (AH). However, validation of these preliminary results is lacking, as well as the impact of HEV infection on the 6-month survival. AIMS: The aims of this study were to evaluate the prevalence of HEV infection markers in an external cohort of patients with histologically proven severe AH and to assess the impact of markers of acute HEV infection on the 6-month survival and the need for liver transplantation (LT). PATIENTS AND METHODS: Patients admitted for severe AH from January 2008 to June 2014 were analysed. HEV serology (IgM and IgG) was retrospectively performed. RESULTS: Ninety-three patients were analysed (male sex 77.4%, age 53±9 years, Maddrey discriminant function 65±32, MELD score 24±6). Six patients (6.5%) had markers of acute HEV infection (IgM+and IgG+), 11 (11.8%) of past HEV infection (IgG+and IgM-) and 76 (81.7%) had a negative serology (IgM- and IgG-). Initial presentation and biological characteristics were not different between IgM+ and IgM- patients, except for the aspartate aminotransferase level (P<0.001). Markers of acute HEV infection had no impact on response to corticosteroids, 1-, 3- or 6-month survival, and the need for LT. Three patients showed symptomatic acute HEV at onset of acute AH: two were treated with ribavirin during the acute phase: one patient died and one patient underwent LT. CONCLUSION: Markers of acute HEV infection were present in 6.5% of patients in our cohort of cirrhotics with histologically proven severe AH, without any impact on short-term or long-term outcome. Whether systematic screening of acute HEV infection in this population should be performed remains an unsolved question.

Coexistence of circulating HBsAg and anti-HBs antibodies in chronic hepatitis B carriers is not a simple analytical artifact and does not influence HBsAg quantification.

BACKGROUND: Presence at the same time of HBsAg and anti-HBs antibodies (HBsAg/Ab) is an entity sometimes encountered in chronic hepatitis B (CHB) carriers. OBJECTIVES: This study was designed to characterize such serological profiles and to assess the reliability of serological marker quantification by three commercially available assays in this setting. STUDY DESIGN: Among 2578 CHB identified patients, 129 (5%) had an HBsAg/Ab profile as determined by Abbott Architect. After exclusion of co-infections (HIV, HCV, HDV), HBV reactivation or HBIg treatment, 101 samples from 62 patients were tested for HBsAg and anti-HBs quantification using Architect, DiaSorin Liaison-XL and Roche Modular-Cobas. Influence of genotype and HBsAg variants was studied in 31 samples with HBV replication. RESULTS: HBsAg detection was confirmed with the 3 techniques for 98% (n = 99) of the samples while the HBsAg/Ab profile was concordant between all techniques for 65% of them. The overall correlation between the 3 HBsAg quantification techniques was good (R(2): 0.94-0.97). The median HBsAg concentration was comparable for the 99 samples whatever the used technique but a bias of -0.11 and 0.02 log IU/mL were noticed for DiaSorin and Roche compared to Abbott, respectively. Anti-HBs quantifications were poorly correlated between techniques with major discrepancies observed. Genotype and substitutions within the "a" determinant showed an impact on HBsAg quantification. CONCLUSIONS: The double HBsAg/Ab profile is not an analytical artifact and is confirmed on all commercially available techniques. While such profile does not influence HBsAg quantification, differences of HBsAg quantification were noticed according to HBV genotype or HBsAg variant.

Performance of the DiaSorin LIAISON(®) anti-HBs II for the detection of hepatitis B surface antibodies: comparison with the Abbott Architect anti-HBs assay.

BACKGROUND: Detection of hepatitis B virus (HBV) surface antibodies (anti-HBs) is required in order to evaluate the response to hepatitis B vaccination and to optimize post-exposure monitoring. The widespread use of vaccines has highlighted the need for accurate and consistent quantification, yielding comparable quantitative results. OBJECTIVES: This study assessed the adequacy of DiaSorin LIAISON(®) anti-HBs II assay in detecting anti-HBs antibodies and determined the correlation with Abbott Architect anti-HBs quantification. STUDY DESIGN: Anti-HBs levels were measured in parallel using Abbott and DiaSorin kits on WHO international standard dilutions and 350 routine samples pre-screened with Abbott Architect anti-HBs. Three different serological panels were tested: vaccinated subjects (n=121); subjects with past HBV infection (n=109); and subjects with no HBV infection marker (n=69). Serial dilutions from nine high-titer anti-HBs samples were used to address linearity. RESULTS: Anti-HBs concentration measured on WHO standard dilutions indicated a good calibration of DiaSorin values against international units, while results for Abbott were below those expected. A strong impact of the dilution matrix used was observed when performing dilutions on high-titer samples. Despite difference in absolute quantification, the overall clinical agreement between the two assays was 96.9%, with strong correlation (r=0.92) between concentrations and good linearity over the quantification range. CONCLUSIONS: The DiaSorin LIAISON(®) anti-HBs II assay shows excellent standardization to the WHO standard and good linearity. The assay is suitable for current clinical laboratory practice.

Impact of hepatitis B virus genotypes and surface antigen variants on the performance of HBV real time PCR quantification.

Quantitative PCR assays used to monitor hepatitis B virus (HBV) load differ in their ability to detect different HBV variants. This study evaluated the performance of the Abbott RT PCR assay for quantitating DNA from different HBV genotypes and from HBV variants bearing HBsAg gene mutations. The study was performed on a randomly-selected sample with a viral load >6logIU/mL for each genotype and on 25 HBsAg variants. Each sample was assayed using the Abbott RT assay and with the Roche Cobas AmpliPrep-Cobas TaqMan as a reference method. All HBV genotypes were detected with the Abbott RT assay with an equivalent dynamic range (1-8logIU/mL). For each genotype, the data suggest that the assay was linear over the entire dilution range (r(2): 0.985-0.995). For the 25 HBsAg variants, viral titres determined with the two assays correlated well (r(2): 0.929). The mean difference between the two methods was -0.295 (95% CI: -0.520 to -0.071). The difference was lower than 1log unit in all but two cases. In conclusion, the Abbott RT assay can detect and quantify DNA from different HBV variants with equivalent performance and is thus suitable for routine monitoring of patients with chronic HBV infections.

A two adenine insertion polymorphism in the 3' untranslated region of factor VII gene is associated with peripheral arterial disease but not with venous thrombosis. Results of case-control studies.

Polymorphic variants of genes encoding blood coagulation proteins have been extensively studied as risk factors for venous or arterial thrombosis A variation in the 3' untranslated region (UTR) involved in the post-transcriptional regulation of factor VII (FVII) gene has been recently identified, a two adenine insertion/deletion at nucleotide 11293. In this study, we investigated its effect on the risk of thrombosis in the frame of two case-control studies, including patients suffering from peripheral arterial disease (PAD) or venous thromboembolic (VTE) disease. The 3'UTR FVII gene polymorphism was investigated i) in 181 patients who had symptomatic atherosclerotic disease of the lower limbs, ii) in 178 patients who had had at least one episode of objectively diagnosed deep venous thrombosis and iii) in controls matched for age and sex. Plasma FVII antigen (FVII: Ag) levels were lower in the presence of the 3'UTR 2A insertion (68.4 +/- 12.3%, 81.3 +/- 14.5% and 89.5 +/- 13.7% in 2A/2A, 2A/0 and 0/0 subjects respectively, p < 0.0001). No significant relationship was found with VTE disease. In the contrary we observed a lower risk of PAD for the 2A/2A compared to the 0/0 genotype after adjustment for traditional risk factors (hypercholesterolemia, smoking status, diabetes and hypertension), with an OR of 0.24 [95% CI 0.06-0.99]. In conclusion, the 2 adenine insertion in the 3'UTR of FVII gene, related to lower plasma FVII levels, is a genetic variation that may contribute to reduce the risk of PAD.

Recurrence of a Phe31Ser mutation in the Gla domain of blood coagulation factor X, in unrelated Algerian families: a founder effect?

The presence of gene lesions in blood coagulation factor X (FX) was investigated in eight FX-deficient patients with severe bleeding symptoms, originating from five unrelated Algerian families (FX coagulant activity <1%, FX antigen ranging from 2% to 16%). A missense mutation (p.Phe31Ser) in the Gla domain was found in homozygous form for all patients but one, who is a compound heterozygote for the Phe31Ser mutation and for a non-sense mutation, Tyr130Term in EGF-2 domain. The haplotypes of FX alleles were determined by the following allelic variants located in the promoter: g.1323_1330delTTGTGA (A1/A2), g.1449T>C, g.1451C>A, upstream to exon 3: g.17257C>T and downstream to exon 3: g.17396A>C. The A1-C-A-T-C haplotype was found on each allele bearing the Phe31Ser mutation in the eight FX deficient patients contrasting with its low frequency (8%) in a control Algerian population (in which the Phe31Ser substitution was absent). The patients came from the same geographical area of Algeria (5/8 are certainly from Kabyle origin) and the haplotype analysis suggests a founder effect. Transient expression study reveals that, for the mutant FX-Phe31Ser, FX antigen level was 60% in conditioned media and 140% in cell lysates compared with the wild type FX. The partial retention and intracellular accumulation of the mutant FX might be due to impaired folding and/or conformational changes, and the discrepancies observed between the FX antigen level in COS-7 cell supernatant (60%) and in the patients plasma (2-16%) to an in vivo increased clearance of the secreted unstable FX mutant.