Abstract Background Although E. coli is generally a well-opted platform for the overproduction of recombinant antigens as heterologous proteins, the optimization of expression conditions to maximize the yield of functional proteins remains empirical. Herein, we developed an optimized E. coli (BL21)-based system for the overproduction of soluble immunoreactive HCV core/envelope proteins that were utilized to establish a novel immunoassay for discrimination of active HCV infection. Methods The core/E1-E2 genes were amplified and expressed in E. coli BL21 (DE3) in the absence/presence of glycylglycine. The antigenic performance of soluble proteins was assessed against 63 HCV-seronegative (Ab − ) sera that included normal and interferent sera (HBV and/or chronic renal failure), and 383 HCV-seropositive (Ab + ) samples that included viremic (chronic/relapsers) and recovered patients’ sera. The color intensity (OD4 50 ) and S/Co values were estimated. Results The integration of 0.1–0.4M glycylglycine in the growth media significantly enhanced the solubility/yield of recombinant core and envelope proteins by ~ 225 and 242 fold, respectively. This was reflected in their immunoreactivity and antigenic performance in the developed immunoassay, where the soluble core/E1/E2 antigen mixture showed 100% accuracy in identifying HCV viremic sera with a viral RNA load as low as 3800 IU/mL, without cross-reactivity against normal/interferent HCV-Ab − sera. The ideal S/Co threshold predicting active viremia (&gt; 2.75) showed an AUC value of 0.9362 (95% CI: 0.9132 to 0.9593), with 87.64, 91.23% sensitivity and specificity, and 94.14, 82.11% positive and negative predictive values, respectively. The different panels of samples assayed with our EIA showed a good concordance with the viral loads and also significant correlations with the golden standards of HCV diagnosis in viremic patients. The performance of the EIA was not affected by the immunocompromised conditions or HBV co-infection. Conclusion The applicability of the proposed platform would extend beyond the reported approach, where glycylglycine, low inducer concentration and post-induction temperature, combined with the moderately-strong constitutive promoter enables the stable production of soluble/active proteins, even those with reported toxicity. Also, the newly developed immunoassay provides a cost-effective point-of-care diagnostic tool for active HCV viremia that could be useful in resource-limited settings. 

Glycylglycine promotes the solubility and antigenic utility of recombinant HCV structural proteins in a point-of-care immunoassay for detection of active viremia

How Frequently Should We Screen for Hepatitis C in US Hemodialysis Centers? Evaluating the Cost-Effectiveness of Different Strategies.

Abstract Background Liver disease assessment is a key aspect of chronic hepatitis C virus (HCV) infection pre-treatment evaluation but guidelines differ on the optimal testing modality given trade-offs in availability and accuracy. We compared clinical outcomes and cost-effectiveness of common fibrosis staging strategies. Methods We simulated adults with chronic HCV receiving care at US health centers through a lifetime microsimulation across five strategies: (1) no staging or treatment (comparator), (2) indirect serum biomarker testing (Fibrosis-4 index [FIB-4]) only, (3) transient elastography (TE) only, (4) staged approach: FIB-4 for all, TE only for intermediate FIB-4 scores (1.45–3.25), and (5) both tests for all. Outcomes included infections cured, cirrhosis cases, liver-related deaths, costs, quality-adjusted life years (QALYs), and incremental cost-effectiveness ratios (ICERs). We used literature-informed loss to follow-up (LTFU) rates and 2021 Medicaid perspective and costs. Results FIB-4 alone generated the best clinical outcomes: 87.7% cured, 8.7% developed cirrhosis, and 4.6% had liver-related deaths. TE strategies cured 58.5%–76.6%, 16.8%–29.4% developed cirrhosis, and 11.6%–22.6% had liver-related deaths. All TE strategies yielded worse clinical outcomes at higher costs per QALY than FIB-4 only, which had an ICER of $12 869 per QALY gained compared with no staging or treatment. LTFU drove these findings: TE strategies were only cost-effective with no LTFU. In a point-of-care HCV test-and-treat scenario, treatment without any staging was most clinically and cost-effective. Conclusions FIB-4 staging alone resulted in optimal clinical outcomes and was cost-effective. Treatment for chronic HCV should not be delayed while awaiting fibrosis staging with TE. 

Clinical- and Cost-Effectiveness of Liver Disease Staging in Hepatitis C Virus Infection: A Microsimulation Study

Significance Statement Studies examining the cost-effectiveness of hepatitis C virus (HCV) screening methods or frequencies are lacking. The authors examined the cost-effectiveness of the Kidney Disease Improving Global Outcomes (KDIGO) 2018 guidelines' recommendation to test in-center hemodialysis patients for HCV every 6 months. They demonstrated that with current HCV prevalence, incidence, and treatment practices in patients receiving hemodialysis, screening for HCV every 6 months is not cost-effective under a willingness-to-pay threshold of US$150,000, even if baseline survival rates doubled or all patients received treatment on diagnosis. Screening only at dialysis initiation or every 2 years are cost-effective approaches, however, with incremental cost-effectiveness ratios of $82,739 and $140,193, respectively, per quality-adjusted life-year saved compared with no screening. These data suggest that reevaluation of HCV screening guidelines in hemodialysis patients should be considered. Background National guidelines recommend twice-yearly hepatitis C virus (HCV) screening for patients receiving in-center hemodialysis. However, studies examining the cost-effectiveness of HCV screening methods or frequencies are lacking. Methods We populated an HCV screening, treatment, and disease microsimulation model with a cohort representative of the US in-center hemodialysis population. Clinical outcomes, costs, and cost-effectiveness of the Kidney Disease Improving Global Outcomes (KDIGO) 2018 guidelines–endorsed HCV screening frequency (every 6 months) were compared with less frequent periodic screening (yearly, every 2 years), screening only at hemodialysis initiation, and no screening. We estimated expected quality-adjusted life-years (QALYs) and incremental cost-effectiveness ratios (ICERs) between each screening strategy and the next less expensive alternative strategy, from a health care sector perspective, in 2019 US dollars. For each strategy, we modeled an HCV outbreak occurring in 1% of centers. In sensitivity analyses, we varied mortality, linkage to HCV cure, screening method (ribonucleic acid versus antibody testing), test sensitivity, HCV infection rates, and outbreak frequencies. Results Screening only at hemodialysis initiation yielded HCV cure rates of 79%, with an ICER of $82,739 per QALY saved compared with no testing. Compared with screening at hemodialysis entry only, screening every 2 years increased cure rates to 88% and decreased liver-related deaths by 52%, with an ICER of $140,193. Screening every 6 months had an ICER of $934,757; in sensitivity analyses using a willingness-to-pay threshold of $150,000 per QALY gained, screening every 6 months was never cost-effective. Conclusions The KDIGO-recommended HCV screening interval (every 6 months) does not seem to be a cost-effective use of health care resources, suggesting that re-evaluation of less-frequent screening strategies should be considered.

A Microsimulation Study of the Cost-Effectiveness of Hepatitis C Virus Screening Frequencies in Hemodialysis Centers

Non-invasive methods have largely replaced biopsy to identify advanced fibrosis in hepatitis C virus (HCV). Guidelines vary regarding testing strategy to balance accuracy, costs and loss to follow-up. Although individual test characteristics are well-described, data comparing the accuracy of using two tests together are limited. We calculated combined test characteristics to determine the utility of combined strategies. This study synthesizes empirical data from fibrosis staging trials and the literature to estimate test characteristics for Fibrosis-4 (FIB4), APRI or a commercial serum panel (FibroSure®), followed by transient elastography (TE) or FibroSure®. We simulated two testing strategies: (1) second test only for those with intermediate first test results (staged approach), and (2) second test for all. We summarized empiric data with multinomial distributions and used this to estimate test characteristics of each strategy on a simulated population of 10,000 individuals with 4.2% cirrhosis prevalence. Negative predictive value (NPV) for cirrhosis from a single test ranged from 98.2% (95% CB 97.6-98.8%) for FIB-4 to 99.4% (95% CB 99.0-99.8%) for TE. Using a staged approach with TE second, sensitivity for cirrhosis rose to 93.3-96.9%, NPV to 99.7-99.8%, while PPV dropped to <32%. Using TE as a second test for all minimally changed estimated test characteristics compared with the staged approach. Combining two non-invasive fibrosis tests barely improves NPV and decreases or does not change PPV compared with a single test, challenging the utility of serial testing modalities. These calculated combined test characteristics can inform best methods to identify advanced fibrosis in various populations.

Test characteristics for combining non-invasive liver fibrosis staging modalities in individuals with Hepatitis C virus.

Care cascade for children with hepatitis C virus exposure in the United States and implications of early ribonucleic acid testing

Negative and positive predictive values of utilizing a two-stage approach to non-invasive fibrosis testing in patients with hepatitis C virus infection

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A Microsimulation Study of the Cost-Effectiveness of Hepatitis C Virus Screening Frequencies in Hemodialysis Centers

Test characteristics for combining non-invasive liver fibrosis staging modalities in individuals with Hepatitis C virus.

Care cascade for children with hepatitis C virus exposure in the United States and implications of early ribonucleic acid testing

Negative and positive predictive values of utilizing a two-stage approach to non-invasive fibrosis testing in patients with hepatitis C virus infection