Insulin resistance is a major determinant of sustained virological response in genotype 1 chronic hepatitis C patients receiving peginterferon alpha-2b plus ribavirin

Background Cross‐sectional studies suggest insulin resistance is strongly associated with hepatic steatosis and fibrosis in patients with chronic hepatitis C (CHC), which might affect the efficacy of antiviral therapy. Aim To investigate retrospectively the impact of insulin resistance on treatment response in Chinese genotype 1 CHC patients receiving a 24‐week course therapy with peginterferon α‐2b/ribavirin. Methods A total of 133 biopsy‐proven CHC patients were enrolled for analyses. Insulin resistance was evaluated by homeostasis model assessment of insulin resistance (HOMA‐IR). Hepatic fibrosis was graded by the METAVIR scoring system. Results Mean HOMA‐IR progressively elevated along with the severity of hepatic fibrosis (F1–F2 fibrosis: 2.55 ± 0.16 vs. F3–F4 fibrosis: 3.61 ± 0.20, P < 0.001). Compared with patients with sustained virological response (SVR), patients without SVR had significantly higher percentages of F3–F4 fibrosis (62.2% vs. 21.6%, P < 0.001) and baseline high viral load (≥600 000 IU/mL; 64.4% vs. 35.6%, P = 0.038). In addition, patients without SVR had significantly higher plasma levels of insulin (15.03 ± 0.89 vs. 10.19 ± 0.55 μU/mL, P < 0.001) and HOMA‐IR values (3.76 ± 0.23 vs. 2.50 ± 0.15, P < 0.001). Multivariate analyses showed that F1–F2 fibrosis (odds ratio: 4.49, P = 0.001), HOMA‐IR < 2 (odds ratio: 7.15, P = 0.005) and pre‐treatment hepatitis C virus RNA < 600 000 IU/mL (odds ratio: 3.26, P = 0.012) were the independent factors associated with SVR. Conclusions Insulin resistance is a major determinant of SVR in genotype 1 CHC patients receiving peginterferon α‐2b/ribavirin. Strategies to modify insulin resistance may be effective in enhancing SVR before or during anti‐viral therapy.     

Hepatic steatosis in chronic hepatitis C: impact on response to anti-viral treatment with peg-interferon and ribavirin

BACKGROUND: There is increasing evidence that hepatic steatosis contributes to the progression of liver fibrosis, whereas its impact on the efficacy of anti-viral treatment is still under investigation. AIM: To evaluate the effect of steatosis on the outcome of combined anti-viral treatment. METHODS: We studied 102 consecutive naive patients with chronic hepatitis C receiving combined anti-viral therapy (peg-interferon alpha-2b and ribavirin). RESULTS: Fifty (49%) of 102 patients had evidence of hepatic steatosis (29 grade 1, 16 grade 2 and 5 grade 3). Sustained virological response was similar in patients with and without steatosis (58% vs. 56%); moreover, the grade of steatosis did not affect the rate of sustained virological response (grade 1: 58%, grade 2: 56% and grade 3: 60%). Patients with steatosis had significantly higher serum levels of aspartate transaminase, alanine transaminase and gamma-glutamyltransferase (P = 0.007, 0.004 and 0.03, respectively), higher histological activity (P = 0.03), more advanced stage of fibrosis (P = 0.0394) and more often hepatitis C virus genotype 3 (P = 0.04). CONCLUSIONS: Our findings suggest that hepatic steatosis in chronic hepatitis C, irrespective of its grade, is not a negative prognostic factor of response to combined anti-viral therapy, even when the histological and biochemical profile of the disease is more aggressive.     

Predicting sustained virological response and anaemia in chronic hepatitis C patients treated with peginterferon alfa-2a (40KD) plus ribavirin

AIM: To assess the likelihood of a sustained virological response (SVR) vs. the likelihood of anaemia in patients with chronic hepatitis C. METHODS: Data from 1732 patients treated with peginterferon alfa-2a (40KD) plus ribavirin in two randomized, multinational studies were pooled. Probabilities of SVR and anaemia were modelled using the generalized additive logistic model, with numerous clinical variables considered for entry into the model. Baseline haemoglobin was only considered in the analysis for anaemia. RESULTS: The probability of anaemia increased from 6 to 16% as a function of the ribavirin dose kg(-1) (12-16 mg kg(-1)), whereas the relationship between SVR and ribavirin dose kg(-1) was influenced by hepatitis C virus (HCV) genotype. The probability of an SVR was not influenced by the ribavirin dose kg(-1) in patients with HCV genotype 2 or 3 infection, but increased as a function of ribavirin dose kg(-1) in patients with HCV genotype 1 infection (40-50% increase in probability of SVR for 12-16 mg kg(-1) dose ribavirin increase). The probability of an SVR in patients included with HCV genotype 1 decreased with increasing HCV RNA level to about 3 million copies ml(-1), but was relatively independent of increasing HCV RNA level thereafter. In addition, older age, a higher ribavirin apparent oral clearance and cirrhosis had a negative impact on achieving an SVR, but improved with increasing alanine aminotransferase (ALT) quotient. Sex and ribavirin dose kg(-1) were the most important prognostic factors for anaemia, followed by baseline haemoglobin, age, baseline ALT quotient and cirrhosis. CONCLUSION: This study supports individualizing ribavirin dosages by HCV genotype and body weight, and highlights several clinical variables that influence the likelihood of an SVR compared with anaemia in chronic hepatitis C patients treated with peginterferon alfa-2a (40KD) plus ribavirin.     

Impact of ribavirin plasma level on sustained virological response in patients treated with pegylated interferon and ribavirin for chronic hepatitis C

Background  The main goal of therapy in hepatitis C virus (HCV) infection is to achieve a sustained virological response (SVR). However, the impact of the pharmacological properties of ribavirin on the SVR has not been fully investigated.
Aim  To evaluate, through a prospective study, the association between ribavirin plasma level and SVR response in HCV patients treated with pegylated interferon (PEG-IFN) and ribavirin.
Patients and methods  Patients treated with PEG-IFN and ribavirin had plasmatic ribavirin dosage at weeks 4 and 12. SVR was evaluated 6 months after the end of treatment.
Results  At week 4, a strong correlation was found between HCV-RNA and C _min of ribavirin plasma level ( r  = −0.376, P  = 0.002) and AUC _0→12h of ribavirin plasma level ( r  = −0.277, P  = 0.018). At week 12, a strong correlation was found between HCV-RNA and C _min of ribavirin plasma level ( r  = −0.384, P  < 0.0001) and AUC _0→12h of ribavirin plasma level ( r  = −0.257, P  = 0.002). In genotype 1 patients, AUC _0→12h ribavirin and C _min were significantly correlated with negative HCV-RNA at week 12 and SVR. In the multiple logistic regression model, the only factor independently associated with SVR in genotype 1 patients was negative HCV-RNA at week 12.
Conclusion  C_min of ribavirin at weeks 4 and 12 was significantly higher in sustained virological responders compared with relapser or nonresponder patients. However, in genotype 1 patients, plasma ribavirin level at weeks 4 and 2 was not associated with SVR.     

Predictive graphical model, network-based medical tool for the prognosis of chronic hepatitis C patients treated with peg-interferon plus ribavirin

Background  There are few model networks to predict treatment outcome in viral hepatitis.
Aim  To develop an easy bioinformatics platform based on algorithm decisions (Bayesian network) for a more efficient prediction of treatment response.
Methods  Totally 385 consecutive chronic hepatitis C (CHC) treated patients were included. More than 40 variables were analysed. Data from 308 patients were used to build the variable model network using DLIFE platform based on predictive graphical models. The prediction accuracy of the bioinformatics network was compared with the true data collected in a retrospective study. The model was then validated twice with external data from CHC patients treated in other hospitals.
Results  The accuracy of this bioinformatics network for treatment response in our 308 patients was 83.3%, which is higher than the accuracy obtained by physicians on the basis of study of clinical data and their own experience (50–65%). The receiver operator characteristic curve areas after validation with another cohort of patients were: 0.91 for sustained virological response, one for nonresponse, and 0.81 for relapse. DLIFE offered a diagnostic accuracy of 81.3%, which is a clear improvement compared with unassisted prognosis (50–65%).
Conclusions  This bioinformatics platform (DLIFE) accurately predicts the outcome of CHC combination therapy, improving treatment decisions and reducing costs. This bioinformatics platform allows integrating widespread data sources and permits predicting the clinical outcome of a particular patient using a general predictive graphical model.     

Cost effectiveness of peginterferon alpha-2a plus ribavirin versus interferon alpha-2b plus ribavirin as initial therapy for treatment-naive chronic hepatitis C

INTRODUCTION: In adults with previously untreated chronic hepatitis C (CHC), the combination of peginterferon alpha-2a plus ribavirin produces a higher rate of sustained virological response (SVR) than interferon alpha-2b plus ribavirin, but it is still unproven whether this increase is cost effective. The objective of this study was to determine if the gain in SVR with peginterferon alpha-2a plus ribavirin is worth the incremental cost. METHODS: We constructed a Markov model of disease progression in which cohorts of patients received peginterferon alpha-2a plus ribavirin or interferon alpha-2b plus ribavirin for 48 weeks (hepatitis C virus [HCV] genotype 1 and non-1 patients with fibrosis) or 24 weeks (genotype non-1 patients without fibrosis), and were followed for their expected lifetimes. The reference patient was a 45-year-old male with CHC without cirrhosis. The SVRs with peginterferon alpha-2a plus ribavirin and interferon alpha-2b plus ribavirin used to populate the model were 46% and 36% for patients infected with HCV genotype 1 and 76% and 61% for patients infected with HCV non-1 genotypes, respectively. QOL and costs for each health state were based on literature estimates and on Italian treatment patterns. Costs were in 2002 euros and benefits were discounted at 3%. Sensitivity analyses on key clinical and economic parameters were performed. The analysis was reported from the perspective of the Italian National Health Service. RESULTS: In patients infected with HCV genotype 1, peginterferon alpha-2a plus ribavirin increased life-years (LYs) by 0.78 years and QALYs by 0.67 years, compared with interferon alpha-2b and ribavirin. The incremental cost per LY and QALY gained was euro9433 and euro10 894, respectively. In patients infected with HCV non-1 genotypes, peginterferon alpha-2a plus ribavirin increased LYs by 1.17 and QALY by 1.01 years, compared with interferon alpha-2b plus ribavirin. The incremental cost per LY and QALY gained was euro3261 and euro3766, respectively. Using genotype distribution estimates, the weighted average ICER for all genotypes was euro6811 per LY gained and euro7865 per QALY gained. CONCLUSION: Our model suggests that peginterferon alpha-2a plus ribavirin is cost effective compared with conventional interferon alpha-2b plus ribavirin for treatment of naive adults with CHC, regardless of HCV genotype, under a wide range of assumptions regarding treatment effectiveness and costs.     

Peginterferon alpha-2a (40kD) [Pegasys] improves HR-QOL outcomes compared with unmodified interferon alpha-2a [Roferon-A]: in patients with chronic hepatitis C

BACKGROUND: Use of unmodified interferon alpha-2a in chronic hepatitis C is associated with impaired health-related quality of life during therapy. Treatment with peginterferon alpha-2a (40kD) provides an improved sustained response over unmodified interferon alpha-2a. OBJECTIVES: To compare health-related quality of life during treatment for patients receiving peginterferon alpha-2a (40kD) [Pegasys] versus unmodified interferon alpha-2a [Roferon]. DESIGN: A randomised, international, multicentre, open-label, parallel group study. SETTING: 36 centres worldwide. PATIENTS: Interferon-na?ve patients (n = 531) with chronic hepatitis C. INTERVENTIONS: Peginterferon alpha-2a (40kD) 180 mirog once a week (n = 267) for 48 weeks or unmodified interferon alpha-2a 6 million IU three times a week for 12 weeks followed by 36 weeks of 3 million IU three times a week (n = 264). MEASUREMENTS: Fatigue Severity Scale and 36-item Short-Form Health Survey (SF-36). RESULTS: At weeks 2 and 12, differences favouring peginterferon alpha-2a (40kD) were seen on seven of eight domains and both summary scores of the SF-36 (p < 0.05 to p < 0.01). At weeks 2, 12 and 24, patients receiving peginterferon alpha-2a (40kD) had less disabling fatigue (p < 0.01) than those receiving unmodified interferon alpha-2a. CONCLUSION: Treatment with peginterferon alpha-2a (40kD) is associated with less disabling fatigue and less impairment in patient functioning and well-being during treatment than unmodified interferon alpha-2a. In addition to safety and efficacy, the impact on health-related quality of life may be an important consideration for physicians when selecting an optimal treatment regimen.     

大剂量利巴韦林联合干扰素治疗快速病毒学应答不佳慢性丙型肝炎的疗效评价

目的:探讨大剂量利巴韦林联合干扰素治疗慢性丙型肝炎快速病毒学应答不佳者的疗效。方法:收集2009年1月至2012年12月收治的132例慢性丙型肝炎快速病毒学应答不佳者,随机分为A、B、C3组,在干扰素治疗的基础上分别给以不同剂量利巴韦林,比较治疗后3组患者早期病毒学应答(early virological response,EVR)、治疗结束时病毒学应答(end of therapyvirological response,ETVR)、持续病毒学应答(sustained virological response,SVR)的差异。结果:治疗后,A组EVR应答26例,ETVR应答33例,SVR应答21例;B组EVR应答19例,ETVR应答26例,SVR应答21例;C组EVR应答14例,ETVR应答17例,SVR应答13例;3组患者在EVR、ETVR和SVR应答率等差异具有统计学意义,大剂量利巴韦林组(A组)疗效明显优于其他组(P<0.05)。结论:对于慢性丙型肝炎快速病毒学应答不佳者,使用大剂量利巴韦林联合干扰素治疗可以提高患者的EVR、ETVR和SVR率。     

Financial impact of two different ways of evaluating early virological response to peginterferon-alpha-2b plus ribavirin therapy in treatment-naive patients with chronic hepatitis C virus genotype 1

BACKGROUND: Patients infected with chronic hepatitis C virus (HCV) genotype 1 are the least responsive to peginterferon (pegIFN) and ribavirin therapy. The monitoring of early virological response (EVR) is therefore an important tool for quickly identifying non-responders, permitting therapy discontinuation and avoiding adverse effects and costs. OBJECTIVE: To analyse the financial impact, in treatment-naive patients infected with HCV genotype 1, of two different measurement techniques for evaluating the EVR during pegIFN-alpha-2b plus ribavirin therapy, and to compare the results of a 48-week standard course of therapy with pegIFN-alpha-2b plus ribavirin without measuring EVR. METHODS: A budget impact model was constructed using a decision-tree analysis. EVR was defined as a >2 log decline in HCV RNA levels at week 12 either tested with two quantitative HCV RNA tests or undetectable HCV core antigen (HCV core Ag) protein levels at week 12 (one HCV core Ag test). Clinical data were taken from multicentre trials and costs from the published literature (euro, 2003 values). The analysis was carried out from the perspective of the Spanish healthcare system and therefore only direct costs were considered. The base-case scenario assumed that a potential study population of 18,504 people in Spain with chronic HCV genotype 1 would be eligible for treatment with pegIFN-alpha-2b plus ribavirin. RESULTS: In the base case, the most effective strategy was testing EVR by HCV core Ag. This resulted in 12,745 patients reaching a sustained virological response (SVR) at an overall cost of 243.98 million euro (19,142 euro per SVR). Conversely, quantitative HCV RNA testing resulted in 11,776 patients with an SVR at a cost of 232.73 million euro ( 19,763 euro per SVR). The incremental cost per successfully treated patient with HCV core Ag testing versus quantitative HCV RNA testing was 11,597 euro. One-way sensitivity analyses demonstrated that changes in the study parameters did not modify the outcomes, except when increasing the EVR or SVR of strategy 2 or when decreasing the EVR or SVR of strategy 3. CONCLUSION: This model suggests, with its underlying assumptions and data, that the assessment of EVR at week 12 by HCV core Ag testing in chronic HCV patients infected with genotype 1 permits identification of those patients expected to achieve an SVR with pegIFN-alpha-2b and ribavirin, resulting in a lower overall cost to the Spanish healthcare system than HCV RNA testing or no testing at all.     

Interferon-beta induction/interferon-alpha2b plus ribavirin therapy in patients with chronic hepatitis C

Treatment of chronic hepatitis C virus (HCV) infection with interferon (IFN) and ribavirin improves the rate of eradication of the virus by less than 20% in patients with genotype 1b and a high viral load. In this study we assessed whether IFN-beta induction/IFN-alpha2b plus ribavirin enhances the efficacy of the therapy in patients with chronic hepatitis C. The efficacy of IFN-beta induction/IFN-alpha2b plus ribavirin therapy (group A, n=7) was compared with that of IFN-alpha2b plus ribavirin (group B, n=7) in 14 patients with high levels of HCV-RNA (> 100 K/U/ml). No significant differences were observed in the clearance of HCV-RNA between the two groups (A and B, respectively) 2 weeks after the start of the treatment (0% and 14.3%), at the end of the treatment (71.4% and 100%) and 6 months after the end of the treatment (28.6% and 14.3%). Recovery was complete in 28.6% and 14.3%, transient in 42.9% and 85.7% and absent in 28.6% and 0% in groups A and B, respectively. Early log changes in the viral load from the baseline after 2 weeks of treatment were 2.41 +/- 0.91 and 2.77 +/- 0.20 in groups A and B, respectively, with no significant difference between the two groups. In the present study, we were not able to demonstrate that IFN-beta induction/IFN-alpha2b plus ribavirin therapy was superior to IFN-alpha2b plus ribavirin therapy in patients with genotype 1b and high viral loads.     

Peginterferon alpha-2b: a new approach to improving response in hepatitis C patients

Chronic hepatitis C infection affects approximately four million Americans. Over the last decade, Type 1 interferons (IFNs) have been the mainstay of therapy for suitable patients. Recently, the combination of IFN plus ribavirin, with enhanced response rates, has replaced IFN monotherapy for treatment of these patients. The addition of a polyethylene glycol (peg) moiety to IFN alpha-2b has provided a drug with reduced clearance whilst retaining biological and antiviral activity. This formulation allows once-weekly dosing and enhances sustained response rates, without significantly changing the safety and tolerability of IFN. Clinical trials indicate a doubling of sustained virological response rates for regimens utilising pegIFN alpha-2b, compared with standard IFN-alpha 2b. The combination of pegIFN alpha-2b and ribavirin further increases the sustained virological response rates to > 50% for suitable patients. Future pegIFN alpha-2b studies will need to examine drug profiles in patients with co-morbid conditions (e.g., renal impairment, liver transplantation), as well as safety and efficacy issues in different ethnic groups. Further clinical trials are also required to determine the benefits of pegIFN alpha-2b in previous non-responders or relapse patients and as maintenance therapy to prevent disease progression. Finally, careful cost effectiveness analyses will need to be performed.     

Sustained virological response to peginterferon plus ribavirin in chronic hepatitis C genotype 1 patients is associated with a persistent Th1 immune response

Summary

Background

An impairment of cellular immune response may contribute to the persistency of hepatitis C virus infection.

Aim

To analyse the Th1/Th2 cytokine profile in peripheral blood CD4+ and CD8+ T cells from patients with chronic hepatitis C (CHC) during treatment with pegylated interferon‐α2a plus ribavirin and to correlate the Th1/Th2 balance with virological response (SVR).

Methods

Prospective longitudinal study: 44 naïve genotype 1 CHC patients received PEG‐IFNα2a plus ribavirin for 48 weeks: 26 (59.1%) achieved a SVR, 13 relapsed (29.5%) and 5 (11.4%) were non‐responders. Sixteen healthy controls were analysed. The production of IL‐4, IFNγ and TNFα by CD4+ and CD8+ T cells was measured using flow cytometry, both in resting and phorbol‐ester‐stimulated cells.

Results

First three months of treatment: the synthesis of TNFα by phorbol‐ester‐stimulated‐CD4+ T cells was higher in patients with SVR (P < 0.01). At the end of treatment, SVR was associated with higher intracellular expression of IFNγ by stimulated‐CD4+ and CD8+ T cells (P < 0.05). At the end of follow‐up, a higher intracellular expression of IFNγ by CD4+ T cells was associated with a SVR.

Conclusions

A Th1‐type immune response was associated with achievement of a SVR, as indicated by the persistent elevation of intracellular IFNγ and TNFα.

     

Assessment of adverse reactions and pharmacokinetics of ribavirin in combination with interferon alpha-2b in patients with chronic hepatitis C

Ribavirin has recently been demonstrated to be efficacious in combination with interferon (IFN) alpha-2b for the treatment of relapsed hepatitis C infections. The aim of this study was to evaluate the relationship between the pharmacokinetics and adverse reactions of ribavirin when ribavirin plus IFN alpha-2b were administered to patients affected with chronic hepatitis C. Nineteen patients received intramuscular IFN alpha-2b at a dose of 6 or 10 million units and oral ribavirin at 600 mg or 800 mg daily for 24 weeks. The pharmacokinetic profiles of ribavirin were assessed by the measurement of plasma concentrations. Twelve patients were continuously given both ribavirin and IFN alpha-2b, whereas in 7 patients the therapy was discontinued due to severe adverse drug reactions such as skin eruption, anemia and depression. There were no significant differences in ribavirin dose, Hb, ALT and AST values between the continued and the discontinued therapy groups. In contrast, the pretreatment platelet level and patient age of the discontinued therapy group were significantly different to the continued group. The trough plasma concentration of ribavirin in the discontinued therapy group was significantly higher than that in the continued group at week 1. These results suggest that the monitoring of plasma ribavirin concentrations may be valid for predicting the early phase of adverse drug reactions, thereby providing useful information for the adjustment of the ribavirin dosing for each patient.     

聚乙二醇干扰素α-2b或联合阿德福韦酯治疗慢性乙型肝炎患者的外周血象变化

目的 观察聚乙二醇干扰素α-2b或联合阿德福韦酯治疗HBeAg阳性慢性乙型肝炎（CHB）患者的外周血象变化。方法 收集2012年6月至2013年9月安徽医科大学第一附属医院、安徽医科大学附属巢湖医院、安徽中医药大学第一附属医院、阜阳市第二人民医院、皖南医学院附属弋矶山医院等10家医院就诊的HBeAg阳性的CHB患者102例,签署知情同意书,随机分为单药组（51例）和联合组（51例）,治疗48周,停药后再随访24周。通过检测受试者基线（0周）、4周、8周、12周、24周、36周、48周、60周、72周的外周血象变化,观察2种治疗方案对外周血象的影响。结果 治疗48周时,联合组患者的HBV DNA转阴率（76.60%）、ALT复常率（70.21%）高于单药组,差异有统计学意义（P<0.05）。不同时间点间白细胞（WBC）、中性粒细胞（N）、淋巴细胞（L）、红细胞（RBC）、血红蛋白（Hb）、血小板（PLT）变化不同,差异有统计学意义（P<0.05）;Hb、PLT与组别之间有交互作用,差异有统计学意义（P<0.05）。单药组对N的影响较联合组更大,差异有统计学意义（P<0.05）。两组患者治疗期间WBC、N、L、PLT均有不同程度的下降,在第0～4周下降最明显,治疗期间趋于稳定,停药后很快恢复正常。结论 联合治疗有一定优势,患者治疗期间外周血WBC、N、L、RBC都具有类似的下降趋势,但联合治疗对Hb、PLT有影响,呈可逆性,停药后很快恢复,治疗期间应严密监测。     

Long-term follow-up of chronic hepatitis C patients with sustained virological response to various forms of interferon-based anti-viral therapy

BACKGROUND: Combination anti-viral therapy achieves a sustained virological response (defined as HCV-RNA negativity 6 months after the end of therapy) of 56% of patients with chronic hepatitis C. Little is known about long-term durability of HCV-RNA negativity in patient treated with pegylated interferon. AIM: To evaluate the durability of virologic response in patients with sustained virological response to anti-viral therapy treated at our centre. METHODS: A total of 187 sustained virological responses (50% genotype 1, 42% genotype 2 or 3 and 8% genotype 4; 20% with cirrhosis) with a follow-up of >12 months post-therapy were studied. Twelve patients received monotherapy with interferon-alpha2a or -2b. One hundred and seventy-five received combination therapy with ribavirin and standard interferon-alpha (n = 73) or pegylated interferon-alpha2a or 2b (n = 102). Qualitative serum HCV-RNA was tested by COBAS AMPLICOR HCV test, v2.0. RESULTS: Median follow-up time was 29 months (range 12-172). Recurrence of HCV infection was not observed in any of the 187 sustained virological responders. Alanine aminotransferase values were normal in 90% and two patients showed minimal elevation of alpha-fetoprotein levels. CONCLUSIONS: No recurrence of HCV infection was seen in any patient. Thus, long-term prognosis in chronic hepatitis C patients with a sustained virological response to therapy with pegylated interferon +/- ribavirin is promising, but long-term studies need to continue.