Prevalence and Pattern of Resistance in NS5A/NS5B in Hepatitis C Chronic Patients Genotype 3 Examined at a Public Health Laboratory in the State of São Paulo, Brazil

Abstract

Purpose Globally, it is estimated that 71 million people are chronically infected with hepatitis C, and 10–20% of these will develop cirrhosis and hepatocellular carcinoma. The development of new direct-acting antiviral (DAA) drugs has contributed to sustained virological response (SVR), eliminating the infection and achieving cure of chronic hepatitis C. However, treated patients can develop HCV resistance to DAAs, which can contribute to the failure of treatment. Here, we aimed to evaluate the prevalence and specific pattern of NS5A and NS5B resistance-associated substitutions (RAS) in samples from patients chronically infected with HCV genotype 3a at a public health laboratory, Instituto Adolfo Lutz, São Paulo, Brazil. Patients and Methods Serum samples from the enrolled individuals were submitted to “in-house” polymerase chain reaction amplification of NS5A and NS5B non-structural protein genes, which were then sequenced by Sanger method. Results A total of 170 and 190 samples were amplified and analyzed for NS5A and NS5B, respectively. For NS5A, 20 (12.0%) samples showed some important RAS; 16 (9.0%) showed some type of substitution and 134 (79.0%) showed no polymorphism. No sample showed any RAS for NS5B. Conclusion This study found important RAS in samples from naïve chronic HCV patients in some areas from São Paulo. The most prevalent were A62S, A30K, and Y93H, which could indicate an increase in resistance to some DAAs used in HCV treatment.

Characterization of Primary Direct Acting Antiviral Drugs (DAA) Resistance Mutations in NS5A/NS5B Regions of Hepatitis C Virus with Genotype 1a and 1b from Patients with Chronic Hepatitis

“…In this study, the Sanger sequencing method was employed for the detection of mutations associated with antiviral drug resistance associated with HCV [15], as a tool with great potential for application in a public health diagnostic laboratory. Many studies on HCV RAS have used this methodology to detect important polymorphisms, as it has a low cost and is easier to perform [13,16,17].…”

“…The population examined represents the patients with most chronic HCV in Brazil who were awaiting treatment. The population was heterogeneous, composed mainly of male individuals who had a high viral load, were approximately 50 years of age and from different regions of the state of São Paulo, Brazil [13,18,19].…”

“…The ampli cation of HCV NS5B region was performed in two steps using the protocol described by Santos et al [13], and a set of primers described in Table 1 [14]. For both regions, ampli ed fragments were visualized by electrophoresis, using 2% agarose gel and SYBR Safe DNA gel stain (Invitrogen TM , Carlsbad, CA, USA).…”

“…Samples were sequenced by Sanger method [15], using commercial Kits, according to the protocol described by Santos et al [13]. To characterize genotypes and identify polymorphisms, the sequences were submitted to Geno2Pheno site (https://hcv.geno2pheno.org).…”

“…Tables Table 1: Sequence of primers used for GT1a and 1b for regions NS5A and NS5B. Adapted from Santos et al [13].…”