Clinical validation of an immunochromatographic SARS-Cov-2 IgM/IgG antibody assay with Japanese cohort.

BACKGROUND: The coronavirus disease 2019 (COVID-19) has become a major health threat. To overcome COVID-19, appropriate diagnosis methods are urgently needed. The aim of this study was to clinically evaluate the colloidal gold immunochromatography assay for SARS-Cov-2 IgM/IgG antibody (Ab). METHODS: Patients confirmed COVID-19 (n = 51) were recruited prospectively from the Musashino Red Cross hospital and Tokyo Medical and Dental University Medical Hospital, between March and May 2020. And the analytical specificity was assessed with serum samples of patients without COVID-19 (n = 100) collected between August to September 2019 before SARS-CoV-2 was first reported in China. RESULTS: Among COVID-19 patients, a total of 87 serum samples were tested for SARS-Cov-2 IgM/IgG Ab assay. IgM was detected 71.0 %, 86.9 %, and 83.3 % at day8-14, 15-28, >29 after symptom onset and IgG was detected in 81.6 %, 87.0 %, and 94.4 %, respectively. The sensitivity of IgM and IgG Ab after day8 assay was significantly higher than before day7, respectively (p=0.0016, 0.0003). There were no positive results in 100 serum samples from patients without COVID-19. CONCLUSION: The SARS-Cov-2 IgM/IgG Ab assay had 79.7% / 86.1% sensitivity (the 8 days after from onset) and 100% specificity in this population.

Genome-wide association study identifies a PSMD3 variant associated with neutropenia in interferon-based therapy for chronic hepatitis C.

Cytopenia during interferon-based (IFN-based) therapy for chronic hepatitis C (CHC) often necessitates reduction of doses of drugs and premature withdrawal from therapy resulting in poor response to treatment. To identify genetic variants associated with IFN-induced neutropenia, we conducted a genome-wide association study (GWAS) in 416 Japanese CHC patients receiving IFN-based therapy. Based on the results, we selected 192 candidate single nucleotide polymorphisms (SNPs) to carry out a replication analysis in an independent set of 404 subjects. The SNP rs2305482, located in the intron region of the PSMD3 gene on chromosome 17, showed a strong association when the results of GWAS and the replication stage were combined (OR = 2.18, P = 3.05 × 10(-7) in the allele frequency model). Logistic regression analysis showed that rs2305482 CC and neutrophil count at baseline were independent predictive factors for IFN-induced neutropenia (OR = 2.497, P = 0.0072 and OR = 0.998, P < 0.0001, respectively). Furthermore, rs2305482 genotype was associated with the doses of pegylated interferon (PEG-IFN) that could be tolerated in hepatitis C virus genotype 1-infected patients treated with PEG-IFN plus ribavirin, but not with treatment efficacy. Our results suggest that genetic testing for this variant might be useful for establishing personalized drug dosing in order to minimize drug-induced adverse events.

Association of gene expression involving innate immunity and genetic variation in interleukin 28B with antiviral response.

UNLABELLED: Innate immunity plays an important role in host antiviral response to hepatitis C viral (HCV) infection. Recently, single nucleotide polymorphisms (SNPs) of IL28B and host response to peginterferon α (PEG-IFNα) and ribavirin (RBV) were shown to be strongly associated. We aimed to determine the gene expression involving innate immunity in IL28B genotypes and elucidate its relation to response to antiviral treatment. We genotyped IL28B SNPs (rs8099917 and rs12979860) in 88 chronic hepatitis C patients treated with PEG-IFNα-2b/RBV and quantified expressions of viral sensors (RIG-I, MDA5, and LGP2), adaptor molecule (IPS-1), related ubiquitin E3-ligase (RNF125), modulators (ISG15 and USP18), and IL28 (IFNλ). Both IL28B SNPs were 100% identical; 54 patients possessed rs8099917 TT/rs12979860 CC (IL28B major patients) and 34 possessed rs8099917 TG/rs12979860 CT (IL28B minor patients). Hepatic expressions of viral sensors and modulators in IL28B minor patients were significantly up-regulated compared with that in IL28B major patients (≈ 3.3-fold, P < 0.001). However, expression of IPS-1 was significantly lower in IL28B minor patients (1.2-fold, P = 0.028). Expressions of viral sensors and modulators were significantly higher in nonvirological responders (NVR) than that in others despite stratification by IL28B genotype (≈ 2.6-fold, P < 0.001). Multivariate and ROC analyses indicated that higher RIG-I and ISG15 expressions and RIG-I/IPS-1 expression ratio were independent factors for NVR. IPS-1 down-regulation in IL28B minor patients was confirmed by western blotting, and the extent of IPS-1 protein cleavage was associated with the variable treatment response. CONCLUSION: Gene expression involving innate immunity is strongly associated with IL28B genotype and response to PEG-IFNα/RBV. Both IL28B minor allele and higher RIG-I and ISG15 expressions and RIG-I/IPS-1 ratio are independent factors for NVR.

Data mining model using simple and readily available factors could identify patients at high risk for hepatocellular carcinoma in chronic hepatitis C.

BACKGROUND & AIMS: Assessment of the risk of hepatocellular carcinoma (HCC) development is essential for formulating personalized surveillance or antiviral treatment plan for chronic hepatitis C. We aimed to build a simple model for the identification of patients at high risk of developing HCC. METHODS: Chronic hepatitis C patients followed for at least 5 years (n=1003) were analyzed by data mining to build a predictive model for HCC development. The model was externally validated using a cohort of 1072 patients (472 with sustained virological response (SVR) and 600 with nonSVR to PEG-interferon plus ribavirin therapy). RESULTS: On the basis of factors such as age, platelet, albumin, and aspartate aminotransferase, the HCC risk prediction model identified subgroups with high-, intermediate-, and low-risk of HCC with a 5-year HCC development rate of 20.9%, 6.3-7.3%, and 0-1.5%, respectively. The reproducibility of the model was confirmed through external validation (r(2)=0.981). The 10-year HCC development rate was also significantly higher in the high-and intermediate-risk group than in the low-risk group (24.5% vs. 4.8%; p<0.0001). In the high-and intermediate-risk group, the incidence of HCC development was significantly reduced in patients with SVR compared to those with nonSVR (5-year rate, 9.5% vs. 4.5%; p=0.040). CONCLUSIONS: The HCC risk prediction model uses simple and readily available factors and identifies patients at a high risk of HCC development. The model allows physicians to identify patients requiring HCC surveillance and those who benefit from IFN therapy to prevent HCC.

Sequences in the interferon sensitivity-determining region and core region of hepatitis C virus impact pretreatment prediction of response to PEG-interferon plus ribavirin: data mining analysis.

The aim of the present study was to clarify the significance of viral factors for pretreatment prediction of sustained virological response to pegylated-interferon (PEG-IFN) plus ribavirin (RBV) therapy for chronic hepatitis C using data mining analysis. Substitutions in the IFN sensitivity-determining region (ISDR) and at position 70 of the HCV core region (Core70) were determined in 505 patients with genotype 1b chronic hepatitis C treated with PEG-IFN plus RBV. Data mining analysis was used to build a predictive model of sustained virological response in patients selected randomly (n = 304). The reproducibility of the model was validated in the remaining 201 patients. Substitutions in ISDR (odds ratio = 9.92, P < 0.0001) and Core70 (odds ratio = 1.92, P = 0.01) predicted sustained virological response independent of other covariates. The decision-tree model revealed that the rate of sustained virological response was highest (83%) in patients with two or more substitutions in ISDR. The overall rate of sustained virological response was 44% in patients with a low number of substitutions in ISDR (0-1) but was 83% in selected subgroups of younger patients (<60 years), wild-type sequence at Core70, and higher level of low-density lipoprotein cholesterol (LDL-C) (≥ 120 mg/dl). Reproducibility of the model was validated (r(2) = 0.94, P < 0.001). In conclusion, substitutions in ISDR and Core70 of HCV are significant predictors of response to PEG-IFN plus RBV therapy. A decision-tree model that includes these viral factors as predictors could identify patients with a high probability of sustained virological response.

Association of IL28B variants with response to pegylated-interferon alpha plus ribavirin combination therapy reveals intersubgenotypic differences between genotypes 2a and 2b.

Genetic polymorphisms of the interleukin 28B (IL28B) locus are associated closely with outcomes of pegylated-interferon (PEG-IFN) plus ribavirin (RBV) combination therapy. The aim of this study was to investigate the relationship between IL28B polymorphism and responses to therapy in patients infected with genotype 2. One hundred twenty-nine chronic hepatitis C patients infected with genotype 2, 77 patients with genotype 2a and 52 patients with genotype 2b, were analyzed. Clinical and laboratory parameters, including genetic variation near the IL28B gene (rs8099917), were assessed. Drug adherence was monitored in each patient. Univariate and multivariate statistical analyses of these parameters and clinical responses were carried out. Univariate analyses showed that a sustained virological response was correlated significantly with IL28B polymorphism, as well as age, white blood cell and neutrophil counts, adherence to RBV, and rapid virological response. Subgroup analysis revealed that patients infected with genotype 2b achieved significantly lower rapid virological response rates than those with genotype 2a. Patients with the IL28B-major allele showed higher virus clearance rates at each time point than those with the IL28B-minor allele, and the differences were more profound in patients infected with genotype 2b than those with genotype 2a. Furthermore, both rapid and sustained virological responses were associated significantly with IL28B alleles in patients with genotype 2b. IL28B polymorphism was predictive of PEG-IFN plus RBV combination treatment outcomes in patients infected with genotype 2 and, especially, with genotype 2b. In conclusion, IL-28B polymorphism affects responses to PEG-IFN-based treatment in difficult-to-treat HCV patients.

Potential relevance of cytoplasmic viral sensors and related regulators involving innate immunity in antiviral response.

BACKGROUND & AIMS: Clinical significance of molecules involving innate immunity in treatment response remains unclear. The aim is to elucidate the mechanisms underlying resistance to antiviral therapy and predictive usefulness of gene quantification in chronic hepatitis C (CH-C). METHODS: We conducted a human study in 74 CH-C patients treated with pegylated interferon alpha-2b and ribavirin and 5 nonviral control patients. Expression of viral sensors, adaptor molecule, related ubiquitin E3-ligase, and modulators were quantified. RESULTS: Hepatic RIG-I, MDA5, LGP2, ISG15, and USP18 in CH-C patients were up-regulated at 2- to 8-fold compared with nonhepatitis C virus patients with a relatively constitutive Cardif. Hepatic RIG-I, MDA5, and LGP2 were significantly up-regulated in nonvirologic responders (NVR) compared with transient (TR) or sustained virologic responders (SVR). Cardif and RNF125 were negatively correlated with RIG-I and significantly suppressed in NVR. Differences among clinical responses in RIG-I/Cardif and RIG-I/RNF125 ratios were conspicuous (NVR/TR/SVR = 1.3:0.6:0.4 and 2.3:1.3:0.8, respectively). Like viral sensors, ISG15 and USP18 were significantly up-regulated in NVR (4-fold and 2.3-fold, respectively). Multivariate and receiver operator characteristic analyses revealed higher RIG-I/Cardif ratio, ISG15, and USP18 predicted NVR. Lower Cardif in NVR was confirmed by its protein level in Western blot. Also, transcriptional responses in peripheral blood mononuclear cells to the therapy were rapid and strong except for Cardif in not only a positive (RIG-I, ISG15, and USP18) but also in a negative regulatory manner (RNF125). CONCLUSIONS: NVR may have adopted a different equilibrium in their innate immune response. High RIG-I/Cardif and RIG-I/RNF125 ratios and ISG15 and USP18 are useful in identifying NVR.

Combinational use of hepatitis B viral antigens predicts responses to nucleos(t)ide analogue/peg-interferon sequential therapy.

BACKGROUND: This prospective cohort study searched for factors associated with a response to nucleos(t)ide analogue/peg-interferon (NUC/peg-IFN) sequential therapy. METHODS: A total of 95 patients with chronic hepatitis B being treated with NUCs were enrolled. Immediately following NUC cessation, peg-IFN was administered at 180 µg/dose weekly for 48 weeks. RESULTS: Twenty-six patients (27%) were judged to be responders at 48 weeks after the completion of peg-IFN. Analysis of baseline factors revealed that hepatitis B surface antigen (HBsAg) <3.1 log IU/ml and HB core-related antigen (HBcrAg) <3.9 log U/ml were significant indicators of a treatment response. The levels of the markers decreased in both responders and non-responders during peg-IFN therapy but continued falling in responders only after halting peg-IFN. Lower HBsAg (<2.0 log IU/ml) and HBcrAg (<3.8 log U/ml) levels at the time of response judgment were also significantly associated with a favorable response. While lower HBcrAg at baseline was the sole predictor of decreased HBcrAg levels at judgment, lower HBsAg, lower HBcrAg, and the use of adefovir dipivoxil at baseline predicted decreased HBsAg levels at the study endpoint. The use of adefovir dipivoxil was also associated with higher serum IFN-λ3, which might have contributed to the reduction in patient HBsAg levels. CONCLUSIONS: The combinational use of HBsAg and HBcrAg levels at baseline and their changes throughout sequential therapy may be useful for predicting a response to NUC/peg-IFN sequential therapy.

Use of simeprevir following pre-emptive pegylated interferon/ribavirin treatment for recurrent hepatitis C in living donor liver transplant recipients: a 12-week pilot study.

BACKGROUND: The management of recurrent hepatitis C following liver transplantation remains a challenge. METHODS: We prospectively investigated the efficacy and safety of simeprevir in combination with pegylated interferon and ribavirin in five patients undergoing living donor liver transplantation (LDLT) with recurrent hepatitis due to hepatitis C virus (HCV) genotype 1b. RESULTS: As the immunosuppressive regimen, four received cyclosporine A (CsA) and one received tacrolimus (FK); no dose adjustment was made prior to the introduction of simeprevir, but the dose was accordingly modified afterwards. All five patients completed the intended 12-week treatment course without significant adverse events greater than grade 2, and no episodes of rejection were detected during the study period. The trough levels of CsA and FK were stably maintained. At week 12, HCV-RNA was not detectable in three of the five patients, whereas the HCV titer of the other two patients, including one with Q80L and V170I mutations at the HCV NS3 position, was at the lower level of quantification (1.2 log10 IU/ml). CONCLUSIONS: Based on this pilot study, simeprevir-based triple therapy is safe and somewhat effective within the first 12 weeks in LDLT recipients with HCV recurrence. Further studies are warranted to obtain robust conclusions.

Association of serum IFN-λ3 with inflammatory and fibrosis markers in patients with chronic hepatitis C virus infection.

BACKGROUND: Hepatitis C virus (HCV) is one of the major causes of liver cancer. The single nucleotide polymorphisms within the IFNL3 gene, which encodes interferon (IFN)-λ(3), are strongly associated with the response to pegylated IFN-α (PEG-IFN-α) plus ribavirin (RBV) therapy in chronic hepatitis C (C-CH) patients. However, the roles of IFN-λ(3) in chronic HCV infection are still elusive. In this study, we aimed to identify clinical and immunological factors influencing IFN-λ(3) and evaluated whether serum IFN-λ(3) levels are involved or not involved in the response to PEG-IFN-α plus RBV therapy. METHODS: We enrolled 119 C-CH patients with HCV genotype 1 infection who underwent 48 weeks of PEG-IFN-α plus RBV therapy. As controls, 23 healthy subjects and 56 patients with non-HCV viral hepatitis were examined. Serum IFN-λ(3) was quantified by chemiluminescence enzyme immunoassay, and 27 cytokines or chemokines were assayed by the multiplexed BioPlex system. RESULTS: Serum IFN-λ(3) levels were higher in C-CH patients or acute hepatitis E patients than in healthy volunteers. Such levels did not differ between the IFNL3 genotypes. In C-CH patients, serum IFN-λ(3) was positively correlated with aspartate aminotransferase, alanine aminotransferase, α-fetoprotein, histological activity, fibrosis index, IFN-γ-inducible protein 10, and platelet-derived growth factor. Multivariate analysis showed that IFNL3 single nucleotide polymorphisms, fibrosis score, and macrophage inflammatory protein 1α were involved in the sustained viral clearance in PEG-IFN-α plus RBV therapy; however, serum IFN-λ(3) levels were not involved. CONCLUSION: Serum IFN-λ(3) levels are increased in C-CH patients regardless of the IFNL3 genotype. IFN-λ(3) is a biomarker reflecting the activity and fibrosis of liver disease, but is not correlated with the responsiveness to PEG-IFN-α plus RBV therapy.

Inhibition of hepatocellular carcinoma by PegIFNα-2a in patients with chronic hepatitis C: a nationwide multicenter cooperative study.

BACKGROUND: We investigated whether the administration of maintenance doses of interferon prevented hepatocellular carcinoma (HCC) in patients with chronic hepatitis C. METHODS: Study 1: A multicenter, retrospective, cooperative study was carried out to determine whether long-term administration of low-dose peginterferon alpha-2a (PegIFNα-2a) prevented HCC development in patients with chronic hepatitis C. In total, 594 chronic hepatitis C patients without a history of HCC were enrolled and treated with 90 µg PegIFNα-2a administered weekly or bi-weekly for at least 1 year. Study 2: HCC developed in 16 of 99 additional patients without PegIFNα-2a treatment during 3.8 years of observation. A propensity-matched control study was then carried out to compare the incidence of HCC between the 59 patients who received low-dose PegIFNα-2a (PegIFNα-2a group) and 59 patients who did not receive PegIFNα-2a treatment (control group), matched for sex, age, platelet count, and total bilirubin levels. RESULTS: Study 1: HCC developed in 49 patients. The risk of HCC was lower in patients with undetectable hepatitis C virus RNA, ≤40 IU/L alanine aminotransferase (ALT), or ≤10 ng/L alpha-fetoprotein (AFP) 24 weeks after the start of therapy. Study 2: The incidence of HCC was significantly lower in the PegIFNα-2a group than in the control group. CONCLUSIONS: Low-dose and long-term maintenance administration of PegIFNα-2a decreased the incidence of HCC in patients with normalized ALT and AFP levels at 24 weeks compared with patients without normal ALT and AFP levels.

Age and total ribavirin dose are independent predictors of relapse after interferon therapy in chronic hepatitis C revealed by data mining analysis.

BACKGROUND: This study aimed to define factors associated with relapse among responders to pegylated interferon (PEG-IFN) plus ribavirin (RBV) therapy in chronic hepatitis C. METHODS: A cohort of genotype 1b chronic hepatitis C patients treated with PEG-IFN plus RBV and who had an undetectable HCV RNA by week 12 (n=951) were randomly assigned to model derivation (n=636) or internal validation (n=315) groups. An independent cohort (n=598) were used for an external validation. A decision tree model for relapse was explored using data mining analysis. RESULTS: The data mining analysis defined five subgroups of patients with variable rates of relapse ranging from 13% to 52%. The reproducibility of the model was confirmed by internal and external validations (r(2)=0.79 and 0.83, respectively). Patients with undetectable HCV RNA at week 4 had the lowest risk of relapse (13%), followed by patients <60 years with undetectable HCV RNA at week 5-12 who received ≥3.0 g/kg of body weight of RBV (16%). Older patients with a total RBV dose <3.0 g/kg had the highest risk of relapse (52%). Higher RBV dose beyond 3.0 g/kg was associated with further decrease of relapse rate among patients <60 years (up to 11%) but not among older patients whose relapse rate remained stable around 30%. CONCLUSIONS: Data mining analysis revealed that time to HCV RNA negativity, age and total RBV dose was associated with relapse. To prevent relapse, ≥3.0 g/kg of RBV should be administered. Higher dose of RBV may be beneficial in patients <60 years.

Pretreatment prediction of response to peginterferon plus ribavirin therapy in genotype 1 chronic hepatitis C using data mining analysis.

BACKGROUND: This study aimed to develop a model for the pre-treatment prediction of sustained virological response (SVR) to peg-interferon plus ribavirin therapy in chronic hepatitis C. METHODS: Data from 800 genotype 1b chronic hepatitis C patients with high viral load (>100,000 IU/ml) treated by peg-interferon plus ribavirin at 6 hospitals in Japan were randomly assigned to a model building (n = 506) or an internal validation (n = 294). Data from 524 patients treated at 29 hospitals in Japan were used for an external validation. Factors predictive of SVR were explored using data mining analysis. RESULTS: Age (<50 years), alpha-fetoprotein (AFP) (<8 ng/mL), platelet count (≥ 120 × 10(9)/l), gamma-glutamyltransferase (GGT) (<40 IU/l), and male gender were used to build the decision tree model, which divided patients into 7 subgroups with variable rates of SVR ranging from 22 to 77%. The reproducibility of the model was confirmed by the internal and external validation (r (2) = 0.92 and 0.93, respectively). When reconstructed into 3 groups, the rate of SVR was 75% for the high probability group, 44% for the intermediate probability group and 23% for the low probability group. Poor adherence to drugs lowered the rate of SVR in the low probability group, but not in the high probability group. CONCLUSIONS: A decision tree model that includes age, gender, AFP, platelet counts, and GGT is useful for predicting the probability of response to therapy with peg-interferon plus ribavirin and has the potential to support clinical decisions regarding the selection of patients for therapy.

Pretreatment prediction of anemia progression by pegylated interferon alpha-2b plus ribavirin combination therapy in chronic hepatitis C infection: decision-tree analysis.

BACKGROUND: This study aimed to develop a model to predict the development of severe anemia during pegylated interferon alpha-2b plus ribavirin combination therapy. METHODS: Data were collected from 1081 genotype 1b chronic hepatitis C patients who were treated at 6 hospitals in Japan. These patients were randomly assigned to a model-building group (n = 691) or an internal validation group (n = 390). Factors predictive of severe anemia (hemoglobin, Hb < 8.5 g/dl) were explored using data-mining analysis. RESULTS: Hb values at baseline, creatinine clearance (Ccr), and an Hb concentration decline by 2 g/dl at week 2 were used to build a decision-tree model, in which the patients were divided into 5 subgroups based on variable rates of severe anemia ranging from 0.4 to 11.8%. The reproducibility of the model was confirmed by the internal validation group (r² = 0.96). The probability of severe anemia was high in patients whose Hb value was <14 g/dl before treatment (6.5%), especially (a) in those whose Ccr was <80 ml/min (11.8%) and (b) those whose Ccr was ≥ 80 ml/min but whose Hb concentration decline at week 2 was ≥ 2 g/dl (11.5%). The probability of severe anemia was low in the other patients (0.4-2.5%). CONCLUSIONS: The decision-tree model that included Hb values at baseline, Ccr, and an Hb concentration decline by 2 g/dl at week 2 was useful for predicting the probability of severe anemia, and has the potential to support clinical decisions regarding early dose reduction of ribavirin.

Relationship between polymorphisms of the inosine triphosphatase gene and anaemia or outcome after treatment with pegylated interferon and ribavirin.

BACKGROUND: A genome-wide association study revealed an association between variants of the inosine triphosphatase (ITPA) gene and ribavirin (RBV)-induced anaemia. The aim of this study was to replicate this finding in an independent Japanese cohort and to define a method to allow pretreatment prediction of anaemia in combination with other factors. METHODS: Genotype 1b chronic hepatitis C patients (n=132) treated with pegylated interferon (PEG-IFN)-α and RBV for 48 weeks were genotyped for ITPA rs1127354 and examined for anaemia and treatment outcome. RESULTS: Variants of the ITPA gene protected against severe anaemia throughout the 48-week treatment period and were associated with lower incidence of anaemia-related RBV dose reduction. A combination of the ITPA genotype with baseline haemoglobin (Hb) and creatinine clearance (CLcr) levels predicted severe anaemia with high accuracy (90% sensitivity and 62% specificity). Among a subset of patients with the IL28B genotype of TT at rs8099917, patients with variants of the ITPA gene were associated with a higher rate of receiving >80% of the expected RBV dose, a higher rate of sustained virological response (SVR), and a lower rate of relapse. CONCLUSIONS: The variants of the ITPA gene, which could protect against haemolytic anaemia and RBV dose reduction, were associated with a high rate of SVR by standard PEG-IFN and RBV therapy in a subset of Japanese patients with the favourable TT genotype at rs8099917 of IL28B. A combination of ITPA genetic polymorphisms with baseline Hb and CLcr levels further improves the predictive accuracy of severe anaemia.