The current standard of treatment for HCV is the combination of pegylated interferon (PEG-IFN) and ribavirin (RBV). Response to therapy is influenced by different factors related to virus or host characteristics. In this study we detected HCV genotype in some patients with chronic HCV infection—who received interferon plus ribavirin therapy and evaluating some risk factors in early resistance to treatment. These risk factors included age, gender, ALT& AST levels, HCV viral load & genotype. This study included 60 patients with chronic HCV infection and subjected to PEG-INF plus RBV therapy. 40 (gp I) had developed resistance after 12 weeks; while group II are the responders. on comparing patients in group I n = 40 (who developed resistance) to patients in group 2, no = 20 (responders), it has been found that the most important risk factors for developing resistance are the increased viral load of HCV-RNA, and AST.HCV-genotype as a risk factor was significantly higher among cases with genotype 1 and 4 and P value was 0.004. This was followed by ALT and AFP as risk factors with P value 0.004 for each and age with P value 0.026. However, regarding sex of the patients there was no significant difference between group I and II. In conclusion: the most frequent HCV genotype in resistant group were genotype I and IV, while in responder patient were genotype 2 & 3. The most important risk factor in this study is viral load and HCV genotype.
The primary goal of treatment is to eradicate (that is/cure) HCV infection [
The current standard of treatment is the combination of pegylated interferon (PEG-IFN) and ribavirin (RBV) [
There are patients who do not achieve a SVR due to unresponsiveness or relapse after treatment, as well as those who lake tolerance to adverse events that occur during treatment. Therefore, the shorter treatment duration is, the more convenient routes of administration and favorable side-effect profiles are [
Response to therapy is influenced by different factors related to virus or host characteristics [
So, our aim in this work is to detect HCV genotype in some patients with chronic HCV infection―who receive interferon plus ribavirin therapy and evaluate predictors of early resistance to treatment.
60 patients were selected for this study infected with HCV and investigated to the treatment of PEG-interferon plus ribavirin. HCV-RNA detected by quantitative real time PCR was performed routinely before treatment and 12 weeks after starting treatment course.
First group: included 40 patients who are non responders to PEG-IFN plus RBV for 12 weeks. Quantitative PCR testing had been performed and proved to be positive.
Second group: is the responders group which included 20 patients who have responded to PEG-IFN plus RBV therapy. Quantitative PCR proved to be negative, six months after completion of treatment course.
All samples were collected after obtaining formal consent from the patients.
5 ml of venous blood was collected from each case under complete aseptic technique and divided into serum and whole blood on EDTA(for platelet count).Serum samples used for HCV genotyping were stored at −20˚C.
Biochemical investigation included ALT and AST levels were determined using automatic autoanalyser, Cobas Amplicor System.
Hematological investigations. Hemoglobin (Hb) measurement and platelets count of the studied samples were performed by automatic system (sysmex).
Determination of degree of fibrosis (F) and degree of activity (A) for a liver biopsy. The activity (A) of the disease which was classified into A0, A1, A2 and A3while degree of fibrosis (F) was classified to F0, F1, F2, F3 and F4 according to the METAVIR classification system [
Hepatic activity score (A)
・ A0 = no activity (no or minimal inflammation).
・ A1 = mild activity.
・ A2 = moderate activity.
・ A3 = severe activity.
Hepatic fibrosis score (F)
・ F0 = no fibrosis (normal liver).
・ F1 = mild fibrosis (portal fibrosis).
・ F2 = moderate fibrosis (few septa).
・ F3 = severe fibrosis (numerous septa).
・ F4 = cirrhosis.
HCV genotyping (this includes viral RNA extraction, RNA purification and cDNA synthesis and amplification).
Viral RNA extraction and purification from the serum samples of patients using a commercial specific kit, Qiaamp viral RNA kit, (QIAGEN GmbH, Germany (www.qiagen.com).
Viral RNA is suspended in 60 ul of buffer AVE equilibrated at room temperature.
cDNA synthesis and amplification: were performed according to manufacturer instructions using commercial Verso 1-Step QRT-PCR ROX KitProvided byThermo FisherScientific (ABgene). ABgene UK. Abgene.techsupport@thermofisher.com. (www.abgene.com).
The sequence of external and internal PCR primers: Specific for 5'-UTR region of HCV genome [
ExF; 5'-AGCGTCTAGCCATGGCGT-3'
ExR; 5'-GCACGGTCTACGAGACCT-3'
InF; 5'-GTGTCTGCGGAACCGG-3'
InR; 5'-GGGCACTCGCAAGCACCC-3'
-The volume of each component in the reaction mix
-QRT-PCR thermal cycling program consist of
Nested-PCR intended to reduce the contamination in products. This involves two sets of primers, used in two successive runs of polymerase chain reaction, the second set intended to amplify a secondary target within the first run product [
3 µl of the first round PCR product was used as template and 0.5 µl of each diluted (1:10) internal primer was added to the reaction. Other PCR components were similar to the first round as follow:
-The volume of each component in the reaction mix
Apart from annealing temperature which was 64˚C for 40 seconds, other steps of nested-PCR program were similar to the first round.
I―Two pairs of restriction enzymes, Hinf I/ScrF I, Hinf I/MvaI and Bsh1236 I, were used and provided by Fermentas life sciences, (www.fermentas.com).
1―HinfI
-Cuts at 5'…G↓A T C…3'
3'…C T A↑G…5'
2―ScrFI
-Cuts at 5'…C C↓ G G…3'
3'…G G ↑C C…5'
3―MvaI
-Cuts at 5'…C C↓ G G…3'
3'…G G ↑C C…5'
4―Bsh1236I
-Cuts at 5'…C G↓C G…3'
3'…G C↑G C…5'
II―1 ml of 10X Fast Digest Buffer: are an advanced line of restriction enzymes for rapid DNA digestion within 5 - 15 minutes.
Pohjanpelto et al. [
・ Protocol for Fast Digestion of PCR product
Sample loading. In each of the three Microtubes (representing one sample) containing DNA & restriction enzymes, 3.0 μl of loading dye was added and Running them on 2% agrose gel: The wells in the gel was loaded with 10.0 μl of 50 bp ladder DNA as well as 10.0 μl of the restriction digest from each Microtube (each microtube represented by a well).
The HCV genotypes were determined in all patients based on PCR-RFLP method.
HCV Genotypes | Size of Fragments | ||
---|---|---|---|
Tube A (ScrF I and Hinf I) | Tube B (Mva I and Hinf I) | Tube C (Bsh1236 I) | |
1a | 97 bp | 97 bp | 129 bp |
1b | 97 bp | 97 bp | 99 bp |
2a | 97 bp | 174 bp | 174 bp |
2b | 174 bp | 174 bp | 174 bp |
3a | 129 bp | 145 bp | 99 bp |
3b | 97 bp | 145 bp | 99 bp |
4 | 97 bp | 145 bp | 129 bp |
5 | 97 bp | 174 bp | 99 bp |
6 | 97 bp | 97 bp | 174 bp |
Group I (n = 40) | Group II (n = 20) | |||
---|---|---|---|---|
Age | 40> | No | 4 | 10 |
% | 10 | 50 | ||
>40 | No | 36 | 10 | |
% | 90 | 50 | ||
Gender | Male | No | 28 | 14 |
% | 70 | 70 | ||
Female | No | 12 | 6 | |
% | 30 | 30 |
Group I (n = 40) | |||||
---|---|---|---|---|---|
P Value | T Value | Mean Change | After Treatment | Before Treatment | Parameters |
Mean ± SD | Mean ± SD | Mean ± SD | |||
0.000 | 4.37 | 1,957,459 ± 2,001,836 | 683,174 ± 105,9010 | 2,640,634 ± 2,354,683 | PCR |
0.000 | −10.27 | −15.35 ± 6.68 | 79.95 ± 11.38 | 64.60 ± 11.53 | ALT |
0.000 | −11.28 | −16.10 ± 6.38 | 89.85 ± 12.69 | 73.75 ± 12.80 | AST |
0.000 | −10.69 | 61,795 ± 25,839 | 120,105 ± 45,030 | 181,900 ± 52,934 | Platelets |
PCR and Platelets were significantly decreased after treatment and P value was 0.000 for each, while ALT and AST were highly significantly increased with P value 0.000 for each.
HCV Genotypes | Group I (n = 40) | Group IV (n = 20) Responders | ||
---|---|---|---|---|
Genotype 1 | 1a | No | 2 | 0 |
% | 5 | 0 | ||
1b | No | 0 | 0 | |
% | 0 | 0 | ||
Genotype 2 | 2a | No | 2 | 0 |
% | 5 | 0 | ||
2b | No | 0 | 0 | |
% | 0 | 0 | ||
Genotype 3 | 3a | No | 0 | 2 |
% | 0 | 10 | ||
3b | No | 8 | 14 | |
% | 20 | 70 | ||
Genotype 4 | No | 28 | 4 | |
% | 70 | 20 | ||
Genotype 5 | No | 0 | 0 | |
% | 0 | 0 | ||
Genotype 6 | No | 0 | 0 | |
% | 0 | 0 |
by 8/40 (20%) cases in group I, and 14/20 (70%) cases in group 2 (responders). Genotype 3a that represented 2/20 (10%) case in group 2 (responders), Genotype 5 and 6 were not found in any of the studied groups.
HCV is a principal cause of chronic liver diseases including liver fibrosis, cirrhosis and hepatocellular carcinoma [
Viral genotypes and viral load are considered the most important predictors of response. The high replication rate of HCV is one of the factors that interfere with the probability of response [
This study included 60 patients with chronic HCV infection and subjected to PEG-INF plus RBV therapy. 40 (gp I) had developed resistance after 12 weeks; they were 28 males and 12 females. Four of them (10%) were less than 40 years and 36 (90%) were more than 40 years.
The comparison between laboratory tests profiles in group I (n = 40) before and after treatment. PCR and platelets were significantly decreased after treatment and P value was 0.000 for each, while ALT and AST were highly significantly increased with P value 0.000 for each (
In similar studies, Alsaran et al., [
P- Value | Odds Ratio (OR) 95% Confidence Interval | Group II (n = 20) Responders | Group I (n = 40) | Risk Factors | |||
---|---|---|---|---|---|---|---|
% | No | % | No | ||||
1.000 | R | 70 | 14 | 70 | 28 | Male | Gender |
1.0 (0.191 - 5.241) | 30 | 6 | 30 | 12 | Female | ||
0.026 | R | 50 | 10 | 10 | 4 | <40 | Age |
9.0 (1.325 - 61.138) | 50 | 10 | 90 | 36 | >40 | ||
0.000 | R | 100 | 20 | 15 | 6 | <600,000 | HCV-PCR |
-- | 0 | 0 | 85 | 34 | >600,000 | ||
0.004 | R | 80 | 16 | 25 | 10 | 2(a&b) + 3(a&b) | HCV-Genotype |
12.0 (1.89 - 76.83) | 20 | 4 | 75 | 30 | 4 + 1(a&b) | ||
0.004 | R | 80 | 16 | 20 | 8 | <55 | ALT |
16.0 (2.39 - 106.73) | 20 | 4 | 80 | 32 | >55 | ||
0.000 | R | 70 | 14 | 5 | 2 | <55 | AST |
44.33 (3.92 - 500.26) | 30 | 6 | 95 | 38 | >55 | ||
0.001 | R | 100 | 20 | 35 | 14 | 1 + 2 | A |
-- | 0 | 0 | 65 | 26 | 3 | ||
0.000 | R | 100 | 20 | 30 | 12 | 1 + 2 | F |
-- | 0 | 0 | 70 | 28 | 3 |
In this study, we performed HCV genotyping for studied patients in order to find a possible correlation between response to treatment and specific genotype. In addition, we tried to discuss the studied clinical and laboratory parameters as risk factors for response versus resistance to IFN therapy plus RBV (
Frequency of different HCV-genotypes is shown in
Lavanchy, [
Several studies have shown that both HCV genotype 2 and 3 are associated with a better response to IFN therapy plus RBV than HCV genotype 1 and 4. HCV genotype 5 was considered to be sensitive to treatment [
So, HCV genotype should be determined before treatment is started. The HCV genotype drives the treatment indication. Patients infected with HCV genotypes other than 1 should be treated with pegylated interferon (PEG-IFN) and RBV only. Patients infected with HCV genotype 1 should receive the triple combination of PEG-IFN, RBV and a protease inhibitor (either telaprevir or boceprevir) [
HCV genotyping is performed by several molecular techniques, such as sequencing of cloned genome, hybridization and Restriction Fragment Length Polymorphism (RFLP). The gold standard method for HCV genotyping is sequencing but this technique is expensive and requires many equipments and facilities, therefore not formidable in most regional laboratories. Contrary, RFLP is a sensitive and cost-effective method especially when a numerous amount of samples need to be genotyped. In RFLP, part of 5’-UTR is amplified by PCR and the amplicon is digested by restriction enzymes. The genotype of the HCV is determined based on the pattern of the fragments following digestion [
Regarding degree of activity as a risk factor for response to treatment, degree of activity (A1 + A2) were found in (35%) patients in non responder group (group I) and (100 %) patients in responders group. (A3) was represented by (65%) of patient in non responder group and was not represented by any patient in responders group (0 %) (
Pockros et al., [
On comparing patients in group I to patients in group II, it has been found that the most important risk factors for developing resistance are the increased HCV viral load, AST level and degree of fibrosis with P value 0.000 for each followed by degree of activity with P value 0.001 (
However, regarding sex of the patients, there was no significant difference between group I and II.
On the contrary, Nachnani et al. [
In a study by Hung et al. [
In conclusion: the most frequent HCV genotype in resistant group were genotype I and IV, while in responder patient were genotype 2 & 3. The most important risk factors in this study were viral load, HCV genotype, degree of activity and fibrosis.