Objectives: The present study aimed to examine the impact of rapid virological response (RVR) and early virological response (EVR) on sustained virological response (SVR) in chronic hepatitis C genotype 3a individuals. Methods: The patients were given antiviral therapy with IFN-α-2b, 3 million units 3 times a week and 800-1,200 mg of ribavirin daily adjusted to the patient's body weight (<60 kg 800 mg day−1, and >60 kg 1,200 day−1). The patients received this combination therapy for 24 weeks. The patients were evaluated for their viral load at week 4, 12, and 24 using RT-PCR. Results: Out of 1,471 patients, 43.3% showed a negative viral load in week 4, demonstrating RVR, whereas 56.6% maintained a high viral load. These were further separated based on viral reduction in their plasma: either negative for HCV-RNA at week 12 (n = 575), manifesting EVR, or showing a 2-log reduction in HCV viral load classified as partial EVR (PEVR; n = 259). The PEVR response was less (29.7%) compared with RVR (85.9%) and EVR (69.0%), although nonresponders were found in both groups. Conclusions: Individuals incompliant with their treatment who have a higher RVR significantly influence their SVR towards a better remission that can be treated within a short duration with standard treatment.

Hepatitis C virus (HCV) is a major cause of liver-associated diseases throughout the world. Approximately 3% of the world's population is chronically infected by HCV, which is due to a high degree of sequence variability in the HCV genome [1]. It is a positive-stranded RNA virus that belongs to the genus Hepacivirus of the Flaviviridae family. The viral infection causes different liver manifestations, such as liver cirrhosis, hepatocellular carcinoma, and certain extrahepatic manifestations (cryoglobulinaemia and sialadenitis). So far, 7 genotypes and 67 subtypes of HCV have been described. Its distribution varies according to geographical location [2].

The current prevalence of HCV infection is very high in Pakistan compared to developed nations due to the lack of healthcare programs [3,4,5]. For example, more than 10 million people were infected with HCV in Pakistan at the start of the 21st century [6,7]. Major causes of HCV transmission are the reuse of syringes and contaminated instruments during surgery and shaving [8]. The HCV pathogenesis is believed to be directly associated with its viral genotype. This assumption is based on the fact that the viral genotype in an individual's blood is associated with treatment outcomes [9]. Genotypic variation is also associated with the geographic distribution of the HCV virus. For example, in Western Europe and North America, HCV types 1a, 1b, 2a, 2b, and 3a are the most prevalent [10], whereas type 4 is endemic to Africa [11]. According to several cross-sectional studies, HCV genotype 3a is more prevalent in Pakistan [12,13]. There have been continuous improvements in the treatment of HCV infection. According to previous studies, combination therapy with interferon (IFN) and ribavirin were used for the treatment of HCV-RNA-positive individuals [14,15,16]. Variability in treatment outcomes is an active area of investigation and involves multiple host factors besides an individual's genotype as well as the viral load [17,18]. In Pakistan, the current standard of care for chronic HCV patients is based on the use of combination therapy involving IFN-α-2b/rIFN-α-2b plus ribavirin, which is the most extensively used antiviral or antineoplastic agent. In cases where a patient is nonresponsive to this therapy, doctors recommend pegylated-IFN combination therapy [19]. We have followed the US Food and Drug Administration predefined criteria (AASLD guidelines) for antiviral administration by giving a combination therapy involving IFN-α-2b/rIFN-α-2b plus ribavirin. NS5A inhibitors (daclatasvir and velpatasvir) and an NS5B inhibitor (sofosbuvir) are used against genotype 3 HCV-infected patients. However, we have used the combination therapy with IFN and ribavirin, which has shown good results and a lower chance of resistance against genotype 3a patients in Pakistan.

A viral response with standard therapeutic treatment differs due to the sustained virological response (SVR) being negative even after 24 weeks of treatment. Therefore, it is essential to carefully consider the use of IFN. The aim of this study was to investigate the predictive value of a rapid virological response (RVR) and early virological response (EVR) on SVR in chronic hepatitis C genotype 3a Pakistani individuals treated with IFN-α-2b plus ribavirin.

Patient Enrolment

HCV-RNA-positive individuals from the peripheries of Islamabad, Pakistan, were enrolled in the present study. Among 5,320 HCV-screened patients, 1,471 were registered at the Nuclear Medicine Oncology and Radiotherapy Institute (NORI) and Pakistan Institute of Medical Sciences (PIMS) in Islamabad between March 2012 and May 2014. The criteria for inclusion are detailed in Table 1. The Ethical Committee of NORI approved the study. Before blood collection, approved informed consent was obtained from patients on a predesigned form in accordance with the country's laws of ethics.

Table 1

Patient inclusion and exclusion criteria

Patient inclusion and exclusion criteria
Patient inclusion and exclusion criteria

Genotyping

The viral genotype was confirmed with routine genotype 3a-specific primers [20,21].

IFN-α-2b plus Ribavirin Treatment

The consenting individuals were given antiviral combination therapy with IFN-α-2b, 3 million units 3 times a week, and 800-1,200 mg of ribavirin daily adjusted according to the patient's body weight (<60 kg 800 mg day−1, and >60 kg 1,200 day−1 with minor modifications based on age and complete blood profile). The participants received this combination therapy for 24 weeks. Their blood samples were collected at week 0, 4, 12, and 24 for evaluation of the HCV viral load. For nonresponders, the therapy was extended to 48 weeks. Patient selection and antiviral administration was in accordance with the American Association for the Study of Liver Diseases (AASLD) practice guidelines [22].

Viral Load Determination

To determine the HCV viral load, RNA was extracted using the automatic RNA extraction unit KingFisher® mL (Thermo Electron Corporation, Vantaa, Finland) with the Instant Virus RNA Extraction Kit (AJ Roboscreen, Berlin, Germany). The detection module of the RoboGene® HCV-RNA Quantification Kit was used to amplify the 5′ untranslated HCV region (5′ UTR) using TaqMan chemistry on a Rotor Gene 3000™ (Corbett Research, Mortlake, Australia). RNA was measured independently for different fluorescence reporter dyes (HCV-RNA: FAM; internal positive control RNA: Yakima Yellow). Data were acquired on FAM and Joe channels. PCR cycling conditions were according to the reported protocol of AJ Roboscreen: step 1: hold 1 at 50°C for 30 min, hold 2 at 95°C for 15 min; cycling (50 repeats), step 2: hold 1 at 95°C for 30 s, hold 2 at 50°C for 60 s, acquiring to cycling A (green, orange); step 3: hold at 72°C for 30 s. The resulting sigmoid growth curves (log scale) were analyzed using the RG software 6.2.25 with a dynamic tube and slope correct settings with a 1% threshold for no template control and a slope value of -3.03 to -3.40 (mean -3.16 ± 0.01), with an R value of 0.9999. The linearity and quantitative range for the assay was 3-5 × 109 copies per run with a detection limit of 3 synthetic HCV-RNA molecules per run. The accuracy and precision of the inter- and intraruns were continuously monitored [23].

Statistical Analysis

The data are presented as percentage frequencies and means ± SD, unless otherwise indicated, using SPSS version 20. A p value <0.05 was considered to be statistically significant. Odds ratios (OR) were calculated where required.

The age of the patients was between 18 and 68 years. Out of 1,471 confirmed genotype 3a individuals, 739 (50.23%) were males and 732 (49.76%) were females. Thepatients were given a combination therapy from the Prime Minister's Program for the Prevention and Control of Hepatitis in Pakistan. In this study cohort, 637 patients achieved RVR (43.3%), 575 (39%) patients had an EVR defined as serum HCV-RNA negativity after 12 weeks of therapy, while 259 (17.6%) patients showed partial EVR (PEVR) with a 2-log drop in HCV-RNA levels (Fig. 1).

Fig. 1

Relative virological responses among the cohort of 1,471 hepatitis C-seropositive individuals based on the predefined criteria of the US Food and Drug Administration.

Fig. 1

Relative virological responses among the cohort of 1,471 hepatitis C-seropositive individuals based on the predefined criteria of the US Food and Drug Administration.

Close modal

The antiviral response rate with combination therapy among patients manifesting an RVR is shown in Figure 2. This group (n = 637) showed a high response rate of 85.9% (n = 545), whereas 14.1% (n = 92) were unresponsive to the treatment. Among 545 responders, SVR was achieved by 96% of the patients (n = 525) and relapse cases were found to be minimal for this category (only 3.1%).

Fig. 2

Longitudinal evaluation of study participants showing RVR, EVR, and PEVR.

Fig. 2

Longitudinal evaluation of study participants showing RVR, EVR, and PEVR.

Close modal

In the EVR patient group (Fig. 2) that took medication for 24 weeks, 69% (n = 397) were declared to be responders. The 31% (n = 178) who were not responsive at the end of treatment were advised by the concerned physician to prolong the medication for a further period of 24 weeks. In EVR patients, among 397 responders, SVR was achieved by 68.5% (n = 272) while 31.5% (n = 125) relapsed. Among these, viral RNA was detected after 6 months of combination therapy. Patients achieving PEVR had lower end treatment response rates (29.7%) and consequently lower rates of SVR (55.8%) as compared to the RVR and EVR groups. The nonresponder patients were advised to undergo a further 24 weeks of treatment (48-week plan). Only 5 patients accepted this option and 4 of them became negative after the completion of the extended treatment plan (Fig. 2). As shown in Table 2, no significant difference of gender was found in EVR subjects; however, the female EVR group was significantly correlated with the early normalization of ALT levels (p < 0.001). According to Table 3, no significant difference was observed in SVR between male (47%) and female (53%) patients (p > 0.05). When the patients were categorized according to viral load, a high percentage of RVR was found in patients with a viral load <100,000 IU/mL as compared to other categories, indicating that as the viral load increases the virological response decreases (Table 4).

Table 2

RVR, EVR, and PEVR to treatment in HCV patients based on gender

RVR, EVR, and PEVR to treatment in HCV patients based on gender
RVR, EVR, and PEVR to treatment in HCV patients based on gender
Table 3

Sustained response to treatment in HCV patients based on gender

Sustained response to treatment in HCV patients based on gender
Sustained response to treatment in HCV patients based on gender
Table 4

Treatment outcomes of patients in relation to viral load

Treatment outcomes of patients in relation to viral load
Treatment outcomes of patients in relation to viral load

On-treatment virological responses were found to be good predictors of achieving a sustained virological response. In RVR patients, there is a 12-fold greater likelihood of SVR (p < 0.000) as compared to EVR cases, while in patients with PEVR there is less chance of SVR as compared to EVR (OR 0.581, p < 0.03). Also, a high probability of SVR was found in RVR patients as compared to non-RVR cases, with a ratio of 1:2. However, the risk of relapse was maximally observed in PEVR (OR 20.756, p < 0.000) cases rather than EVR cases (OR 1.721 with 95% CI 1.047-2.829, p < 0.03), while keeping the RVR as reference.

The recorded data on side effects among the patients showed that weakness (27%, n = 394) and fatigue (24%, n = 356) were experienced by most of the subjects. Loss of hair and symptoms of fever were recorded in 17 and 19% of the patients, respectively, with most of the hair loss observed in females (58%, n = 149) below the age of 30 years. Anxiety and general body ache was observed in 3% (n = 43) and 10% (n = 141) of the cases, respectively. Vision problems were recorded in elderly subjects. Almost 49% of the patients (n = 715) tolerated the medication well, with no notable side effects. However, most of these were below 30 years of age, implying some side effects may be age related. A visit to the dentist was found to be the most common route of infection (32%), while a significant number of patients (45%) were unaware of the possible cause of infection.

Multiple studies have confirmed the prevalence rate of genotype 3 as 60-90% in Pakistan [24,25,26,27,28,29,30]. This wide range of occurrence clearly indicates the population-specific genotype distribution of HCV [31]. The current study was conducted in an institute catering for patients from different cities of Pakistan, including Gujranwala, Mianwali, Peshawar, Sakardu, Rawalpindi, and Islamabad. The treatment with IFN and ribavirin is effective in approximately 80% of patients with HCV genotype 2 or 3 infection [32].

Monitoring of the level of RVR and EVR until the end of treatment as well as the SVR is crucial in the management of chronic HCV, not only to achieve a cure but also as an indicator of acquiescence and adherence to therapy. Being a very poor country, Pakistan cannot afford expensive treatment plans. Conventional durations of therapy for chronic HCV patients can potentially be made cost effective, with little or no exposure to therapy side effects and even enhanced rates of SVR. According to Dutch national guidelines for the treatment of chronic HCV infection [33], RVR may allow the shortening of therapy for genotypes 3. In contrast to the findings of Stroffolini et al. [34], we did not find a significant SVR difference between male (47%) and female (53%) patients (p > 0.05).

In the current study, the RVR group exhibited a good end of treatment response (ETR) when compared with the EVR and PEVR groups. A high rate of SVR (96.3%) was achieved in the RVR group as compared with the recent study conducted by Idrees and Riazuddin [35] on HCV genotype 3 in Pakistani patients with IFN-α-2b combination therapy, who reported an SVR rate of more than 85% in RVR, suggesting that patients with RVR had a significantly higher SVR. Other studies support these findings by suggesting high rates of SVR among patients with an RVR within 16-24 weeks of therapy [36]. Patients exhibiting PEVR have shown lower rates of ETR followed by low rates of SVR. These RVR, EVR, and PEVR categories can be used as a relapse risk predictor. SVR is more expected in the RVR category (p < 0.001), whereas the maximum relapse risk was observed in the PEVR category, with a ratio of 2:1 (p < 0.001) versus and EVR ratio of 1:7 (p < 0.03). EVR has limited value as a predictor of response in genotype 3 patients as it is achieved by almost 98% of the cases. However, RVR measured at week 4 is a strong predictor of SVR, which was achieved by 96.0% of patients (n = 525). This outcome was consistent with other reported studies [20] and patients achieving RVR may be suitable candidates for shorter treatment durations, minimizing the economic burden.

Viral kinetics during therapy provides information on how to individualize treatment. Patients with RVR and EVR had excellent SVR rates, ranging from 76 to 84% regardless of the treatment plan (48 vs. 72 weeks), whereas patients with no EVR had higher SVR rates of 72 instead of 48 weeks (29 vs. 17%, p = 0.040) [37]. In the current study, extension of the treatment plan in 11 patients (from 24 to 32 or 48 weeks) showed promising results. Five patients who showed an RVR were declared as nonresponders at the end of 24 weeks of treatment. Four patients responded to medication up until 32 weeks, which was also supported by a previous study that reported the effect of treatment extension with pegylated-IFN-α-2a plus ribavirin from 48 to 72 weeks [38]. The treatment significantly increased the rate of SVR in patients with detectable viremia at the 4th week of treatment. In the absence of EVR and no ETR, patients showed negative RNA levels with an extended combination therapy plan of 48 weeks. Nonresponders with PEVR might benefit from an extended course of therapy; however, this approach needs to be validated prospectively [39]. This information can be taken as supporting evidence for treating patients.

In summary, RVR was found to be a strong predictor of SVR, whereas failure to achieve EVR is a strong predictor of an unsustained virological response, independent of the patient's pretreatment status. The combination of RVR and EVR provided consistent data and thus offers a key to individualizing treatment, as well as a basis for advocating adherence to therapy.

The combination therapy involving IFN-α-2b/rIFN-α-2b plus ribavirin showed better results in the chronic HCV genotype 3a patients in Pakistan. However, there is a need for individual treatment and potential treatment algorithm modifications. Extended treatment plans can be advised to selected individuals to achieve a better SVR.

We highly appreciate the provision of laboratory facilities by the Nuclear Medicine Oncology and Radiotherapy Institute (NORI) in Islamabad. The personal assistance of I.A. by KFUPM is acknowledged.

The authors declare that they have no conflicts of interest.

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