NS5A sequence heterogeneity of hepatitis C virus genotype 4a predicts clinical outcome of pegylated-interferon-ribavirin therapy in Egyptian patients

Abstract

Hepatitis C virus genotype 4 (HCV-4) is the cause of approximately 20% of the 180 million cases of chronic hepatitis C in the world. HCV-4 infection is common in the Middle East and Africa, with an extraordinarily high prevalence in Egypt. Viral genetic polymorphisms, especially within core and NS5A regions, have been implicated in influencing the response to pegylated-interferon and ribavirin (PEG-IFN/RBV) combination therapy in HCV-1 infection. However, this has not been confirmed in HCV-4 infection. Here, we investigated the impact of heterogeneity of NS5A and core proteins of HCV-4, mostly subtype HCV-4a, on the clinical outcomes of 43 Egyptian patients treated with PEG-IFN/RBV. Sliding window analysis over the carboxy terminus of NS5A protein identified the IFN/RBV resistance-determining region (IRRDR) as the most prominent region associated with sustained virological response (SVR). Indeed, 21 (84%) of 25 patients with SVR, but only 5 (28%) of 18 patients with non-SVR, were infected with HCV having IRRDR with 4 or more mutations (IRRDR ≥ 4) (P = 0.0004). Multivariate analysis identified IRRDR ≥ 4 as an independent SVR predictor. The positive predictive value of IRRDR ≥ 4 for SVR was 81% (21/26; P = 0.002), while its negative predictive value for non-SVR was 76% (13/17; P = 0.02). On the other hand, there was no significant correlation between core protein polymorphisms, either at residue 70 or at residue 91, and treatment outcome. In conclusion, the present results demonstrate for the first time that IRRDR ≥ 4, a viral genetic heterogeneity, would be a useful predictive marker for SVR in HCV-4 infection when treated with PEG-IFN/RBV.

Fig 1

Sliding window analysis over the carboxy terminus (aa 2193 to 2417) of NS5A of HCV-4 obtained from SVR and non-SVR patients.

Fig 2

Correlation between IRRDR[HCV-4] sequence variations and treatment outcome. (A) Average number of amino acid mutations in IRRDR[HCV-4] obtained from SVR and non-SVR patients. (B) Alignment of IRRDR[HCV-4] sequences obtained from SVR and non-SVR patients with HCV-4a, -4m, -4n, and -4o. The consensus sequence (Cons) of each subtype is shown on the top. The numbers along the sequence indicate the amino acid positions. Dots indicate residues identical to those of the Cons sequence. The numbers of the mutations in each IRRDR (4a, 4m, 4n, or 4o) are shown on the right.

Fig 3

ROC curve analysis of IRRDR[HCV-4] sequence heterogeneity for SVR prediction. The solid line curve shows the AUC. Solid circles with numerals plotted on the curve represent different numbers of IRRDR mutations analyzed. The dashed line in the upper left corner indicates the optimal number of IRRDR[HCV-4] mutations for SVR prediction, which yields the highest sensitivity (84%) and the highest specificity (72%).

Fig 4

Sequence alignment of the core protein of HCV-4 isolates. Core protein sequences (aa 1 to 120) of HCV-4 obtained from SVR and non-SVR patients are aligned. The prototype sequence of ED43 (10) is shown on the top. The numbers along the sequence indicate the amino acid positions. Dots indicate residues identical to those of the prototype sequence.