Advertisement

A method for rapid detection and genotype identification of hepatitis C virus 1–6 by one-step reverse transcription loop-mediated isothermal amplification

Open AccessPublished:December 11, 2015DOI:https://doi.org/10.1016/j.ijid.2015.12.002

      Highlights

      • Rapid detection of hepatitis C virus (HCV) genotypes 1–6.
      • HCV detection in heat-treated plasma without RNA extraction.
      • Field-deployable and point-of-care HCV assay.
      • HCV detection assay for resource-limited settings.

      Summary

      Objective

      Hepatitis C virus (HCV) is probably the leading cause of liver cirrhosis and hepatocellular carcinoma globally. Diagnostic tools conventionally used for the detection and identification of HCV infection are technically demanding, time-consuming, and costly for resource-limited environments. This study reports the development of the first rapid loop-mediated reverse transcription isothermal amplification assay that rapidly detects and identifies HCV genotypes in blood components.

      Methods

      RNA extracted from donor plasma and serum specimens was applied to a one-step reverse transcription loop-mediated isothermal amplification reaction performed with HCV-specific oligonucleotides. Reactions were conducted at 63.5 °C for 30–60 min. The diagnostic characteristics of the assay were investigated and validated with clinical specimens.

      Results

      Electrophoretic analysis of amplification revealed detection and identification of HCV genotypes 1–6. Positive amplification revealed unique ladder-like banding patterns that identified each HCV genotype. The assay demonstrated a sensitivity of 91.5% and specificity of 100%. Rapid naked-eye detection of HCV infection was facilitated by observation of an intense fluorescent glow of amplified targets under UV illumination.

      Conclusion

      These diagnostic characteristics highlight the potential utility of this assay for the rapid detection and genotype identification of HCV infection in field and point-of-care settings in endemic regions and resource-limited environments.

      Graphical abstract

      Keywords

      1. Introduction

      Hepatitis C virus (HCV) is a single-stranded RNA virus of the Flaviviridae family.
      • Moratorio G.
      • Martínez M.
      • Gutiérrez M.F.
      • González K.
      • Colina R.
      • López-Tort F.
      • et al.
      Evolution of naturally occurring 5′ non-coding region variants of hepatitis C virus in human populations of the South American region.
      Transmitted through modes including injection drug use, contaminated needle-stick injuries, and unsafe blood transfusion, infection with HCV may lead to chronic active hepatitis and hepatocellular carcinoma.
      • Ghany M.G.
      • Strader D.B.
      • Thomas D.L.
      • Seeff L.B.
      Diagnosis, management, and treatment of hepatitis C: an update.
      • Liang T.J.
      • Rehermann B.
      • Seef L.B.
      • Hoofnagel J.H.
      Pathogenesis, Natural History, Treatment, and Prevention of Hepatitis C.
      NIH consensus statement on management of hepatitis C: 2002.
      Approximately 185 million people are infected with HCV worldwide, with developing countries of Sub-Saharan Africa, Asia, North and South America, and the Middle East most affected.
      • Messina J.P.
      • Humphreys I.
      • Flaxman A.
      • Brown A.
      • Cooke G.S.
      • Pybus O.G.
      • et al.
      Global distribution and prevalence of hepatitis C virus genotypes.
      • Gower E.
      • Estes C.
      • Blach S.
      • Razavi-Shearer K.
      • Razavi H.
      Global epidemiology and genotype distribution of the hepatitis C virus infection.
      • Zein N.
      Clinical significance of hepatitis C virus genotype.
      There are seven major genotypes of HCV with 67 subtypes found in different regions of the world.
      • Smith D.B.
      • Bukh J.
      • Kuiken C.
      • Muerhoff A.S.
      • Rice C.M.
      • Stapleton J.T.
      • et al.
      Expanded classification of hepatitis C virus into 7 genotypes and 67 subtypes: united criteria and genotype assignment Web resource.
      • Lamballerie X.
      • Charrel R.N.
      • Attoui A.H.
      • De Micco P.
      Classification of hepatitis C virus variants in six major types based on analysis of the envelope 1 and nonstructural 5B genome regions and complete polyprotein sequences.
      • Simmonds P.
      • Alberti A.
      • Alter H.J.
      • Bonino F.
      • Bradley D.W.
      • Brechot C.
      • et al.
      A proposed system for the nomenclature of hepatitis C viral genotypes.
      Globally, HCV genotype 1 is the most common, accounting for about 46% of all infections. This is followed by genotype 3, accounting for 22% of the global HCV burden, and genotypes 2 and 4, each accounting for 13%.
      • Messina J.P.
      • Humphreys I.
      • Flaxman A.
      • Brown A.
      • Cooke G.S.
      • Pybus O.G.
      • et al.
      Global distribution and prevalence of hepatitis C virus genotypes.
      • Gower E.
      • Estes C.
      • Blach S.
      • Razavi-Shearer K.
      • Razavi H.
      Global epidemiology and genotype distribution of the hepatitis C virus infection.
      • Zein N.
      Clinical significance of hepatitis C virus genotype.
      The detection of HCV infection in blood derivatives and the identification of the genotypes are therefore important in clinical diagnostics and antiviral treatment, ensuring blood safety, and providing epidemiological information about HCV prevalence.

      Infectious Diseases Society of America (IDSA). Recommendations for testing, managing and treatment of hepatitis C. IDSA; 2014. Available at: www.hcvguidelines.org (Accessed date: November 3, 2015).

      • De Leuw P.
      • Sarrazin C.
      • Zeuzem S.
      How to use virological tools for the optimal management of chronic hepatitis C.
      • Etoh R.
      • Imazeki F.
      • Kurihara T.
      • Fukai K.
      • Fujiwara K.
      • Arai M.
      • et al.
      Pegylated interferon-alfa-2a monotherapy in patients infected with HCV genotype 2 and importance of rapid virological response.
      A plethora of molecular diagnostic methods have been designed and used for the detection and genotyping of HCV infection. While these tests are highly sensitive, they remain expensive and laborious and require well-trained personnel, as well as sophisticated laboratory facilities.
      • Rho J.
      • Ryu J.S.
      • Hur W.
      • Kim C.W.
      • Jang J.W.
      • Bae S.H.
      • et al.
      Hepatitis C virus (HCV) genotyping by annealing reverse transcription-PCR products with genotype-specific capture probes.
      • Nolte F.S.
      • Thurmond C.
      • Fried F.W.
      Preclinical evaluation of AMPLICOR hepatitis C virus test for detection of hepatitis C virus RNA.
      • Sábato M.F.
      • Shiffman M.L.
      • Langley M.R.
      • Wilkinson D.S.
      • Ferreira-Gonzalez A.
      Comparison of performance characteristics of three real-time reverse transcription-PCR test systems for detection and quantification of hepatitis C virus.
      • Duarte C.A.
      • Foti L.
      • Nakatani S.M.
      • Riediger I.N.
      • Poersch C.O.
      • Pavoni D.P.
      • et al.
      A novel hepatitis C virus genotyping method based on liquid microarray.
      Furthermore, the application of these tests may be limited regarding their ability to detect and simultaneously identify the specific HCV genotypes.
      • De Keukeleire S.
      • Descheemaeker P.
      • Reynders M.
      Diagnosis of hepatitis C virus genotype 2k/1b needs NS5B sequencing.
      Besides, several (reverse transcription) loop-mediated isothermal amplification (RT)-LAMP assays have been designed for the detection of various pathogens including HCV, but they detect a limited number of genotypes and rarely demonstrate genotype identification of the pathogens targeted for detection.
      • Notomi T.
      • Okayama H.
      • Masubuchi H.
      • Yonekawa T.
      • Watanabe K.
      • Amino N.
      • et al.
      Loop-mediated isothermal amplification of DNA.
      • Nagamine K.
      • Hasse T.
      • Notomi T.
      Accelerated reaction by loop-mediated isothermal amplification using loop primers.
      • Blomström A.L.
      • Hakhverdyan M.
      • Reid S.M.
      • Dukes J.P.
      • King D.P.
      • Belák S.
      • et al.
      A one-step reverse transcriptase loop-mediated isothermal amplification assay for simple and rapid detection of swine vesicular disease virus.
      • Kargar M.
      • Askari A.
      • Doosti A.
      • Ghorbani-Dalini S.
      Loop-mediated isothermal amplification assay for rapid detection of hepatitis C virus.
      • Yang J.
      • Fang M.X.
      • Li J.
      • Lou G.Q.
      • Lu H.J.
      • Wu N.P.
      Detection of hepatitis C virus by an improved loop-mediated isothermal amplification assay.
      • Wang Q.Q.
      • Zhang J.
      • Hu J.S.
      • Chen H.T.
      • Du L.
      • Wu L.Q.
      • et al.
      Rapid detection of hepatitis C virus RNA by a reverse transcription loop-mediated isothermal amplification assay.
      • Sun B.
      • Rodriguez-Manzano J.
      • Selck D.A.
      • Khorosheva E.
      • Karymov M.A.
      • Ismagilov R.F.
      Measuring fate and rate of single-molecule competition of amplification and restriction digestion, and its use for rapid genotyping tested with hepatitis C viral RNA.
      Several reports have been published regarding RT-LAMP assays that have utilized the HCV 5′ non-coding region (5′-NCR) for primer design and the detection of HCV-RNA.
      • Kargar M.
      • Askari A.
      • Doosti A.
      • Ghorbani-Dalini S.
      Loop-mediated isothermal amplification assay for rapid detection of hepatitis C virus.
      • Yang J.
      • Fang M.X.
      • Li J.
      • Lou G.Q.
      • Lu H.J.
      • Wu N.P.
      Detection of hepatitis C virus by an improved loop-mediated isothermal amplification assay.
      • Wang Q.Q.
      • Zhang J.
      • Hu J.S.
      • Chen H.T.
      • Du L.
      • Wu L.Q.
      • et al.
      Rapid detection of hepatitis C virus RNA by a reverse transcription loop-mediated isothermal amplification assay.
      • Sun B.
      • Rodriguez-Manzano J.
      • Selck D.A.
      • Khorosheva E.
      • Karymov M.A.
      • Ismagilov R.F.
      Measuring fate and rate of single-molecule competition of amplification and restriction digestion, and its use for rapid genotyping tested with hepatitis C viral RNA.
      Unfortunately, these works have been limited to detection only and have demonstrated no pattern formation of the bands, thereby hindering the clear determination of true positive detection, contamination, or cross-reactivity. Also, RT-LAMP assays traditionally rely on electrophoretic gel end-point analysis of banding patterns to determine positive amplification. Therefore, it is important that laddering of RT-LAMP amplicons is arranged in clear and distinct patterns to enable easy analysis and interpretation of amplification results.
      This study reports the development of the first RT-LAMP method for the detection and simultaneous genotype identification of HCV genotypes 1–6. The assay is simple, sensitive, and performed on the molecular basis of auto cycling strand-displacement DNA synthesis, which produces long stem-loop products of multiple inverted repeats under isothermal amplification.
      • Notomi T.
      • Okayama H.
      • Masubuchi H.
      • Yonekawa T.
      • Watanabe K.
      • Amino N.
      • et al.
      Loop-mediated isothermal amplification of DNA.
      • Nagamine K.
      • Hasse T.
      • Notomi T.
      Accelerated reaction by loop-mediated isothermal amplification using loop primers.
      • Blomström A.L.
      • Hakhverdyan M.
      • Reid S.M.
      • Dukes J.P.
      • King D.P.
      • Belák S.
      • et al.
      A one-step reverse transcriptase loop-mediated isothermal amplification assay for simple and rapid detection of swine vesicular disease virus.
      The amplification process is rapid and accomplished in less than 60 min, utilizing two thermostable enzymes and two to three sets of oligonucleotides. The oligonucleotides target conserved as well as sparsely polymorphic sequences in the 5′-NCR of the HCV genome
      • Bukh J.
      • Purcell R.H.
      • Miller R.H.
      Sequence analysis of the 5′ noncoding region of hepatitis C virus.
      (Table 1), producing distinctly clear banding patterns that indicate positive detection for possible target identification. In this study, a new approach to HCV detection and genotype identification is introduced and discussed.
      Table 1Oligonucleotide sequences and color-matched illustration of set DN3 primers on the targeted HCV 5′-NCR
      HCV, hepatitis C virus; NCR, non-coding region.
      GenBank accession numbers of HCV 5′-NCR used for primer design: DQ480524a, Y13184b, and AF009606c.

      2. Materials and methods

      2.1 Nucleic acid standards and plasma panels

      Quantified Armored RNA standards of HCV1a, HCV1b, and HCV2ac, HIV-1B, dengue virus (DENV) 1, and West Nile virus (WNV) were purchased from Asuragen (Austin, TX, USA). HCV genotyping plasma panels of World Health Organization (WHO) International Standard were used in the development of the assay. The panels included HCV Worldwide AccuSet Performance Panel 0810-0173 (SeraCare, Milford, MA, USA), Worldwide HCV Performance Panel WWHV303 (SeraCare, USA), and AcroMetrix HCV Genotyping Panel (Applied Biosystems/Life Technologies, Grand Island, NY, USA). Also, the hepatitis B virus (HBV) plasma panel of WHO International Standard (Applied Biosystems/Life Technologies, USA) was used as the control in the development of the assay.

      2.2 Design of oligonucleotide sequences

      Full-length sequences of various HCV genotypes were obtained from the GenBank database of the National Center for Biotechnology Information (NCBI) and analyzed using CLUSTAL-W2. Primer sets targeting the 5′-NCR of the HCV genome were designed manually and consisted of the following: forward inner primer (FIP), reverse inner primer (RIP), loop forward primer (LF), loop reverse primer (LR), forward outer primer (F3), and reverse outer primer (R3). A ‘TTTT’ spacer was used to link the two sequences of FIP (F1c and F2) and RIP (R1c and R2). The primers targeted conserved as well as sparsely polymorphic nucleotide sequences in the HCV 5′-NCR. Several sets of primers were designed and analyzed, and three sets designated as DN1, DN2, and DN3 were used in this assay (Table 1). The oligonucleotides were synthesized by Eurofins MWG Operon (Huntsville, AL, USA) and Integrated DNA Technologies (Coralville, IA, USA).

      2.3 Specimens and nucleic acid extraction

      A total of 171 archival blood donor clinical specimens were tested in order to evaluate the clinical applicability of the test. Total RNA was extracted from healthy human plasma specimens (n = 100) and clinical donor plasma specimens infected by HCV (n = 65) that were pre-selected using the US Food and Drug Administration (FDA)-approved Procleix test
      • Ginocchio C.C.
      Life beyond PCR: alternative target amplification technologies for the diagnosis of infectious diseases, part II.
      (packet insert) for comparison. RNA was also isolated from HCV-3a clinical plasma samples (n = 3; kindly provided by Dr J. Stapleton), HCV-4a clinical serum specimens (n = 3; kind gift from Dr M. Ghany), and from the HCV genotyping plasma panels. Extraction of RNA was performed with the QiaAmp Viral RNA Mini Kit protocol (Qiagen, Germantown, MD, USA) with some modifications, as follows: approximately 200 μl of plasma and serum were used for RNA extraction, while 4 μl of RNA-Secure (Ambion/Life Technologies, Grand Island, NY, USA) were added to the 60-μl RNA elution to protect the nucleic acid from degradation. Viral DNA, used as assay control, was extracted from the HBV plasma panel (Applied Biosystems/Life Technologies, USA) using the QiaAmp DNA Mini Kit (Qiagen, USA) according to the manufacturer's protocol. The eluted nucleic acids were aliquoted and stored at −80 °C until needed for testing.

      2.4 HCV diagnostic genotyping assay

      Detection and identification of HCV was performed by loop-mediated reverse transcription isothermal amplification in a 25-μl total reaction mixture. The mixture comprised 12.5 μl of 2× mannitol acetate buffer (MAB),
      • Nyan D.C.
      • Ulitzky L.E.
      • Cehan N.
      • Williamson P.
      • Winkelman V.
      • Rios M.
      • et al.
      Rapid detection of hepatitis B virus in blood plasma by a specific and sensitive loop-mediated isothermal amplification assay.
      1 μM each of primers FIP and RIP, 0.6 μM each of primers LF and LR, 0.5 μM each of primers F3 and R3, 8 U of Bst DNA polymerase (New England Biolabs, MA, USA), 5 U of cloned AMV reverse transcriptase, and 10 U of RNaseOUT (Invitrogen,, MD, USA). An RNA template volume of 5 μl was applied to the reaction. A no-template (water) control was included in all experiments. Positive controls included known genotypes of HCV-RNA standards. HIV, DENV, WNV, HBV DNA (OptiQuant-AcroMetrix/Life Technologies, Benicia, CA, USA), and normal human plasma were used as assay negative controls. Reactions were performed at 63.5 °C for 30–60 min on a portable digital heat-block, the MyBlock Mini Dry Bath (Benchmark Scientific, Edison, NJ, USA), and terminated by placing the reaction tubes on ice.

      2.5 Analysis of amplicons

      Reaction products were analyzed by running 5 μl of amplification products on a 2.8% agarose gel prepared in 1× TBE (89 mM Tris-base, 89 mM boric acid, 2 mM ethylenediaminetetraacetic acid (EDTA); pH 8.0) and stained with GelRed (Phenix Research Products, Candler, NC, USA). Products were electrophoresed for 50–55 min at 120 volts in 1× TBE buffer, visualized under a UV transilluminator at 302 nm, and photographed with the G:BOX gel documentation system (Syngene, Frederick, MD, USA). For rapid acquisition of results, 0.5 μl of 10× GelGreen dye (Phenix Research Products, USA) was added to 10 μl of amplification products in the tubes. The tubes were then visualized under a UV transilluminator at 302 nm and photographed with an iPad-Air tablet camera (Apple Inc., Cupertino, CA, USA) or a BlackBerry Z30 smartphone camera (Research-In-Motion, Ontario, Canada). Electrophoretic analysis of banding patterns and visual interpretation of fluorescence intensity in the reaction tubes were performed by at least three laboratory personnel.

      2.6 Specificity and sensitivity studies

      Assay specificity and the cross-reactivity of primers were evaluated by testing the HCV oligonucleotides against nucleic acids of HIV, DENV, WNV, and HBV. Primer sets were also evaluated for their ability to produce banding patterns common to all HCV genotypes tested, or to produce distinguishable ladder-like banding patterns unique to each HCV genotype. Sensitivity and the limit of detection (LOD) of the RT-LAMP genotyping assay were evaluated by testing serial dilutions of quantitated HCV-RNA ranging from 105 to 0.1 IU per reaction (IU/rxn). A probit analysis was also conducted by testing 5–6 replicates of the HCV-RNA dilutions (105 to 0.1 IU/rxn).

      2.7 Assay inhibition studies

      The detection of HCV-RNA was performed in the presence of nucleic acids of other pathogens in order to investigate the ability of the assay to detect its target without inhibition by co-purified nucleic acid substrates. Hence, the target HCV-RNA (103 IU/rxn) was amplified in an RT-LAMP reaction in the presence of either HIV-RNA (106 IU/rxn) or HBV-DNA (105 IU/rxn), and with both HIV and HBV nucleic acids combined.

      2.8 Time-course of HCV-RNA detection

      In order to determine the time-point at which HCV-RNA was amplified, time-course experiments were conducted by testing two higher dilutions of HCV-RNA (10 and 50 IU/rxn) at defined reaction time intervals (20, 30, 40, and 60 min). Negative control reactions ran for 60 min.

      2.9 Evaluation of assay with donor specimens

      The clinical application of the HCV RT-LAMP genotyping assay was evaluated by testing clinical specimens infected by HCV (n = 71) and normal/healthy human plasma specimens (n = 100). Total RNA was extracted from the specimens and 5–10 μl of RNA was subjected to isothermal amplification. Reaction products were resolved on a 2.8% agarose gel to analyze the resulting banding patterns that distinguish the HCV genotypes.

      2.10 Detection of HCV-RNA with heat-treated plasma specimens

      In order to evaluate the ability of the assay to amplify viral nucleic acid without RNA isolation, 50 μl of standard quantitated plasma specimens (OptiQuant-AcroMetrix/Life Technologies) were serially diluted with normal human plasma. The dilutions were heated for approximately 5–10 min at 33.5 °C. Next, 5 μl of heat-treated materials ranging from 106 to 102 IU were applied directly to the isothermal amplification reaction for the detection of HCV-RNA.

      3. Results

      3.1 Detection and analysis of products

      Total RNA extracts of HCV genotypes 1–6 were subjected to isothermal amplification. Electrophoretic analysis of reaction products demonstrated positive amplification of HCV-RNA by primer sets DN1 and DN2. The oligonucleotides produced a ladder-like banding pattern common to HCV genotypes 1–6, while the assay reaction was negative to DENV, WNV, HIV, and HBV, as confirmed by the absence of a banding pattern (Figure 1A ; Supplementary Material Figure S1A). For rapid naked-eye visualization of the results, GelGreen DNA intercalating dye was added to the reaction tubes, which revealed an intense fluorescence glow in tubes with amplified RNA as compared with the no-template control (NTC), DENV, WNV, HIV, and HBV (Figure 1B; Supplementary Material Figure S1B).
      Figure thumbnail gr1
      Figure 1Detection, specificity, and identification of HCV genotypes. (A) Results of electrophoresis showing positive amplification of HCV genotypes 1–6 utilizing oligonucleotide set DN1, but no detection of dengue virus (lane D) or West Nile virus (lane W). The results demonstrate the presence of similar ladder-like banding patterns in HCV genotypes 1–6, but the absence of banding pattern in the no-template control (NTC), D, and W. (B) Rapid detection of targets by UV visualization of amplified targets (HCV 1–6) demonstrated by an intense fluorescent glow as compared with the negative controls. (C) Duplicates of HCV genotypes 1, 3, 4, 5, and 6 were subjected to isothermal amplification using primer set DN3. Electrophoretic analysis showed the detection of all targets with differentiating ladder-like banding patterns unique to each detected genotype, as illustrated by the differentiating color lines. M = 100 bp marker; NTC = non-template control; NC = negative control; D = dengue virus; W = West Nile virus.

      3.2 Specificity and genotype identification

      The assay specificity and cross-reactivity of the primers were evaluated by amplification and gel electrophoretic analysis. As observed on the gels, the primers detected only HCV-RNA, but reacted negative to nucleic acids of DENV (or D), WNV (or W), HIV, and HBV (Figure 1A; Supplementary Material Figure S1). Interestingly, primer set DN3 reacted positive to HCV genotypes 1, 3, 4, 5, and 6, producing banding patterns of amplicons that were unique to each genotype detected, although weakly positive for HCV-3 (Figure 1C). Differences in banding patterns were identified by keen observation of the pattern locations relative to the molecular marker, size of the bands within the patterns, spacing of each group of patterns, and the laddering-shift of the patterns, as illustrated by the color lines between the duplicate samples (Figure 1C). When clinical donor specimens were tested using primer set DN3, the results revealed detection of HCV-1 and HCV-6 (Figure 2; Supplementary Material Figure S2 and Table S1).
      Figure thumbnail gr2
      Figure 2Diagnostic specificity and identification of HCV genotypes in clinical specimens. In order to evaluate the clinical utility of the assay, total RNA extracted from clinical specimens was subjected to isothermal amplification utilizing primer set DN3. Electrophoretic analysis of the amplified products demonstrated the detection of HCV-1 (lane 3) and HCV-6 (lane 5) with distinct ladder-like banding patterns. Note the differentiation in banding pattern (aided by illustrative color dots). Lane 1 = NTC (no template control); lane 2 = PC (positive control HCV-1); lane 3 = HCV-1 clinical specimen; lane 4 = HCV-5 positive control; lane 5 = HCV-6 clinical specimen; M = 100-bp marker; lanes 6–9 = NP (normal plasma specimens).

      3.3 Assay sensitivity

      The assay sensitivity and LOD were determined by testing serial dilutions of HCV-RNA (105–0.1 IU/rxn). The results of electrophoretic analysis demonstrated detection of 10 IU/rxn of HCV-RNA (Figure 3A) . The addition of GelGreen fluorescent dye to the reaction tubes revealed a fluorescent glow of decreasing intensity that corresponded to the level of HCV-RNA amplified (Figure 3B). Probit test of HCV-RNA replicates demonstrated a 100% detection rate for 105, 104, 103, and 102 IU, an 80% detection rate for 50 IU, and a 40% detection rate for 10 IU; 1 IU and 0.1 IU of HCV-RNA were not detected (Table 2A) .
      Figure thumbnail gr3
      Figure 3Assay detection sensitivity. Sensitivity was evaluated using serial dilutions of HCV-RNA. The assay detected down to 10 IU/rxn of RNA (lane 8) and demonstrated fluorescence of decreasing intensity corresponding to the amount of nucleic acid detected (lanes 3–8). NTC = no-template control; NC = negative plasma control; M = 100-bp marker; HCV = hepatitis C virus.
      Table 2Evaluation of assay diagnostic sensitivity and specificity
      A. Probit data of replicates of HCV-RNA serial dilutions
      HCV-RNA dilutions (IU)Number of replicates testedNumber of positive reactionsPercent positive

      (%)
      10566100
      10455100
      10355100
      10255100
      505480
      105240
      1.0500
      0.1500
      B. Test of clinical specimens
      HCV-positive specimensHCV-negative specimensTotal tested
      Positive test65065
      Negative test6100106
      Total specimens71100171
      C. Diagnostic test evaluation
      Value95% CI
      Sensitivity (%)91.582.5–96.8
      Specificity (%)10096.3–100
      Positive predictive value (%)10094.4–100
      Negative predictive value (%)94.388.0–97.8
      Positive likelihood ratio00
      Negative likelihood ratio0.080.04–0.18
      HCV, hepatitis C virus; CI, confidence interval.

      3.4 Detection interference studies

      The ability of the assay to specifically detect HCV nucleic acid in the presence of non-target nucleic acids was evaluated. Agarose gel analysis of the reaction products revealed no inhibition of HCV-RNA amplification, as demonstrated by the presence of ladder-like banding patterns in lanes 5, 6, and 7; the resulting banding patterns were similar to banding patterns observed in the HCV-positive control in lane 4 (Figure 4). No banding pattern was observed in the HIV-RNA and HBV-DNA negative control reactions in lanes 1, 2, and 3, respectively (Figure 4).
      Figure thumbnail gr4
      Figure 4Detection of HCV in the presence of other viruses. HCV-RNA was tested against the background of HIV and HBV nucleic acids. Electrophoretic results of amplification showed the detection of HCV-RNA in lanes 5–7 in the presence of HIV-RNA and HBV-DNA; this is confirmed by the presence of ladder-like banding patterns identical to the HCV-positive control in lane 4 and the absence of banding pattern in lanes 1–3. NTC = no-template control; HIV = human immunodeficiency virus (106 IU/rxn); HBV = hepatitis B virus (105 IU/rxn); HCV = hepatitis C virus (103 IU/rxn); M = 100-bp marker.

      3.5 Time-course of detection

      In order to determine the time-point at which amplification of HCV-RNA occurred, 10 and 50 IU of RNA were tested in the isothermal amplification reaction at designated time-points. Results of the electrophoretic analysis of reaction products revealed amplification of 10 IU/rxn of RNA at 60 min, while amplification of 50 IU/rxn was observed at 40 min (Figure 5).
      Figure thumbnail gr5
      Figure 5Assay time-point of HCV-RNA detection. The time-point at which detection occurs was evaluated using 50 and 10 IU/rxn of HCV-RNA in the amplification reaction at defined time intervals over a 60-min period. Electrophoretic analysis demonstrated the detection of 50 IU of HCV-RNA at the 40-min time-point, while 10 IU/rxn was detected at the 60-min time-point. NTC = no-template control; M = 100 bp marker.

      3.6 Detection and identification of HCV in clinical specimens

      In order to determine the clinical applicability of the RT-LAMP genotyping assay, total RNA was extracted from 171 clinical donor specimens and tested using primer set DN3. Of the 71 HCV-positive clinical donor specimens tested, the assay detected a total of 65 HCV-infected specimens: 58 plasma specimen reactions were positive for HCV-1, two plasma specimens tested positive for HCV-6, two of the known HCV-3 serum specimens tested positive, and the three known HCV-4 plasma specimens also tested positive, while a total of six HCV-infected specimens reacted negative. All healthy human plasma specimens (n = 100) tested negative (Figure 2, Table 2B; Supplementary Material Figure S2 and Table S1). As presented in Table 2C, the assay demonstrated a diagnostic sensitivity of 91.5% with a confidence interval (CI) of 82.5–96.8%, while the specificity was 100% with a 95% CI of 96.3–100%.

      3.7 Detection of HCV-RNA with heat-treated plasma specimens

      The assay was also investigated for its ability to amplify viral nucleic acid from heat-treated specimens without RNA extraction. When serial dilutions of heated plasma specimens were tested in the amplification reactions, electrophoretic results demonstrated the detection of HCV-RNA (106–104 IU/rxn), as confirmed by the presence of banding patterns (Supplementary Material Figure S3).

      4. Discussion

      HCV is a major health care problem worldwide. The detection of HCV and genotype identification provide important insights into the clinical progression of liver disease, the response to antiviral therapies, and the dynamics of the HCV global epidemiological profile. This study demonstrated the performance of a specific and simple isothermal amplification assay for the rapid detection and genotype identification of HCV infection in plasma and serum. This work describes the first RT-LAMP assay that detects six HCV genotypes using single primer sets such as DN1 and DN2. This assay demonstrates advantages over labor-intensive methods that require heavy equipment and multiple diagnostic steps for HCV detection and genotype identification.
      • Albertoni G.
      • Arnoni C.P.
      • Araújo P.R.
      • Carvalho F.O.
      • Barreto J.A.
      Signal to cut-off (S/CO) ratio and detection of HCV genotype 1 by real-time PCR one-step method: is there any direct relationship?.
      • Detmer J.
      • Arnoni C.P.
      • Araújo P.R.
      • Carvalho F.O.
      • Barreto J.A.
      Accurate quantification of hepatitis C virus (HCV) RNA from all HCV genotypes by using branched-DNA technology.
      • Kotwal G.J.
      • Baroudy B.M.
      • Kuramoto I.K.
      • McDonald F.F.
      • Schiff G.M.
      • Holland P.V.
      • et al.
      Detection of acute hepatitis C virus infection by ELISA using a synthetic peptide comprising a structural epitope.
      • Hara K.
      • Rivera M.M.
      • Koh C.
      • Sakiani S.
      • Hoofnagle J.H.
      • Heller T.
      Important factors in reliable determination of hepatitis C virus genotype by use of the 5′ untranslated region.
      • Shemis M.A.
      • El-Abd D.M.
      • Ramadan D.I.
      • El-Sayed M.I.
      • Guirgis B.S.
      • Saber M.A.
      • et al.
      Evaluation of nested polymerase chain reaction for routine hepatitis virus genotyping in Egyptian patients.
      Conducted as a one-step procedure, this assay also obviates the need for extra cDNA synthesis and restriction digest steps for genotype identification. In this assay, both the synthesis and amplification of HCV-RNA were performed in a single reaction tube, using a single temperature. Additionally, the assay detected and distinguished the HCV genotypes without requiring post-amplification genotyping procedures such as restriction enzyme analysis, reverse hybridization, or nested RT-PCR.
      • Sun B.
      • Rodriguez-Manzano J.
      • Selck D.A.
      • Khorosheva E.
      • Karymov M.A.
      • Ismagilov R.F.
      Measuring fate and rate of single-molecule competition of amplification and restriction digestion, and its use for rapid genotyping tested with hepatitis C viral RNA.
      • Albertoni G.
      • Arnoni C.P.
      • Araújo P.R.
      • Carvalho F.O.
      • Barreto J.A.
      Signal to cut-off (S/CO) ratio and detection of HCV genotype 1 by real-time PCR one-step method: is there any direct relationship?.
      • Detmer J.
      • Arnoni C.P.
      • Araújo P.R.
      • Carvalho F.O.
      • Barreto J.A.
      Accurate quantification of hepatitis C virus (HCV) RNA from all HCV genotypes by using branched-DNA technology.
      • Kotwal G.J.
      • Baroudy B.M.
      • Kuramoto I.K.
      • McDonald F.F.
      • Schiff G.M.
      • Holland P.V.
      • et al.
      Detection of acute hepatitis C virus infection by ELISA using a synthetic peptide comprising a structural epitope.
      • Hara K.
      • Rivera M.M.
      • Koh C.
      • Sakiani S.
      • Hoofnagle J.H.
      • Heller T.
      Important factors in reliable determination of hepatitis C virus genotype by use of the 5′ untranslated region.
      • Shemis M.A.
      • El-Abd D.M.
      • Ramadan D.I.
      • El-Sayed M.I.
      • Guirgis B.S.
      • Saber M.A.
      • et al.
      Evaluation of nested polymerase chain reaction for routine hepatitis virus genotyping in Egyptian patients.
      Moreover, this assay successfully amplified HCV-RNA utilizing heated specimens without conventional RNA extraction (Supplementary Material Figure S3). This method of substrate preparation enhances the rapidity of detection and may provide a cost-saving approach for laboratories in resource-limited settings. Notwithstanding its time-saving nature and cost-effectiveness, further improvements to this method of template preparation are required.
      HCV circulates in various geographic regions of the world with high prevalence.
      • Smith D.B.
      • Bukh J.
      • Kuiken C.
      • Muerhoff A.S.
      • Rice C.M.
      • Stapleton J.T.
      • et al.
      Expanded classification of hepatitis C virus into 7 genotypes and 67 subtypes: united criteria and genotype assignment Web resource.
      • Lamballerie X.
      • Charrel R.N.
      • Attoui A.H.
      • De Micco P.
      Classification of hepatitis C virus variants in six major types based on analysis of the envelope 1 and nonstructural 5B genome regions and complete polyprotein sequences.
      • Simmonds P.
      • Alberti A.
      • Alter H.J.
      • Bonino F.
      • Bradley D.W.
      • Brechot C.
      • et al.
      A proposed system for the nomenclature of hepatitis C viral genotypes.
      Thus in most developed countries, blood donor screening for HCV infection and surveillance are performed using state-of-the-art tools for early diagnosis to ensure the safety of the blood supply and to monitor the epidemiological distribution pattern of the virus. In contrast, this poses a challenge in developing countries where diagnostic resources are limited or unavailable. With its demonstrated diagnostic characteristics, the RT-LAMP assay reported in this study may thus contribute to addressing the diagnostic needs of resource-limited environments and underserved communities (Table 2).
      The HCV assay demonstrated a sensitivity of approximately 10 IU/rxn of extracted RNA (Figure 3), making it potentially capable of early diagnosis of HCV infection. Also, the assay revealed a good analytical specificity without cross-reacting with other viral pathogens, as the assay detection of HCV-RNA was not inhibited by the presence of HBV or HIV nucleic acids (Figure 4). Moreover, the assay detected HCV-RNA in 65 of 71 infected clinical specimens, thus revealing a 91.5% diagnostic sensitivity (Table 2C; Supplementary Material Table S1). Also, the probit data showed a 100% detection rate for 102 IU and an 80% detection rate for 50 IU of HCV-RNA at the lower dynamic range of the assay (Table 2A). These results are in agreement with previously published reports of (RT)-LAMP sensitivity and specificity.
      • Blomström A.L.
      • Hakhverdyan M.
      • Reid S.M.
      • Dukes J.P.
      • King D.P.
      • Belák S.
      • et al.
      A one-step reverse transcriptase loop-mediated isothermal amplification assay for simple and rapid detection of swine vesicular disease virus.
      • Wang Q.Q.
      • Zhang J.
      • Hu J.S.
      • Chen H.T.
      • Du L.
      • Wu L.Q.
      • et al.
      Rapid detection of hepatitis C virus RNA by a reverse transcription loop-mediated isothermal amplification assay.
      • Nyan D.C.
      • Ulitzky L.E.
      • Cehan N.
      • Williamson P.
      • Winkelman V.
      • Rios M.
      • et al.
      Rapid detection of hepatitis B virus in blood plasma by a specific and sensitive loop-mediated isothermal amplification assay.
      This work utilized the 5′-NCR of the HCV genome for primer design due to its conserved nature across HCV genotypes, thus facilitating the detection of all HCV genotypes tested. This work also exploited the sparse nucleotide polymorphism present within the HCV 5′-NCR for genotype identification, as revealed by the electrophoretic differences in banding patterns of the detected targets (Figure 1C). When evaluated with clinical donor specimens, the test identified specimens that were positive for HCV genotypes 1 and 6 (Figure 2; Supplementary Material Figure S2 and Table S1). Collectively considered, these results have demonstrated the potential capability of this isothermal assay for specific detection and identification of HCV genotypes. Although a demonstrated proof of concept, this assay would benefit from further evaluation in a field setting with a larger number of clinical samples in order to improve its diagnostic and analytical characteristics.
      A unique characteristic of this RT-LAMP genotyping assay is its high specificity, not only due to the absence of cross-reactivity with other pathogens, but by its demonstration of clearly distinct electrophoretic laddering patterns of the targeted HCV genotypes. It was consistently observed that the loop primers play a critical role in banding pattern formation (data not shown – to be published elsewhere) and primarily generate the resulting differences in laddering patterns. This study therefore postulates that the loop primers may have utilized the sparsely existing nucleotide diversity within the conserved 5′-NCR to generate the differences observed in laddering pattern (Figure 1C, Figure 2; Supplementary Material Figure S1).
      The role of genotypes in the progression of HCV infection and liver diseases is well documented. For example, HCV genotypes 1b and 3 are reportedly associated with more severe liver diseases, while the HCV response to antiviral therapies may also vary by genotype.
      • Nkontchou G.
      • Ziol M.
      • Aout M.
      • Lhabadie M.
      • Baazia Y.
      • Mahmoudi A.
      • et al.
      HCV genotype 3 is associated with a higher hepatocellular carcinoma incidence in patients with ongoing viral C cirrhosis.
      • Bruno S.
      • Silini E.
      • Crosignani A.
      • Borzio F.
      • Leandro G.
      Hepatitis C virus genotypes and risk of hepatocellular carcinoma in cirrhosis: a prospective study.
      • El-Shamy A.
      • Hotta H.
      Impact of hepatitis C virus heterogeneity on interferon sensitivity: an overview.
      • Sherman K.E.
      • Flamm S.L.
      • Afdhal N.H.
      • Nelson D.R.
      • Sulkowski M.S.
      • Everson G.T.
      • et al.
      Response-guided telaprevir combination treatment for hepatitis C virus infection.
      • Maekawa S.
      • Enomoto N.
      Viral factors influencing the response to the combination therapy of peginterferon plus ribavirin in chronic hepatitis C.
      This highlights the therapeutic implications of genotype as a critical factor when implementing individualized therapies. With the exception of HCV-2 and HCV-7 RNA standards, which were unavailable at the time of further investigation, this assay successfully distinguished several HCV genotypes utilizing the electrophoretic banding pattern (Figure 1C). Also, besides testing HCV-1a and 1b, this assay did not investigate different subtypes of the other genotypes tested due to the unavailability of said test materials. Notwithstanding this drawback, this simple diagnostic method may empower health care providers delivering services in underserved environments, mainly in developing countries; the demonstrated rapidity of the assay, amplifying approximately 50 IU of HCV-RNA in less than 60 min, may contribute to timely clinical decision-making (Figure 5). However, additional studies including field trials are warranted for further evaluation and improvement of the diagnostic capabilities of this assay.
      In conclusion, the data presented in this study demonstrate a reverse transcription isothermal amplification assay with the capability of detecting HCV infections at the genotypic level. Due to its sensitivity, specificity, and lack of requirements for expensive equipment, this assay is potentially suitable for field and point-of-care use in resource-limited settings and HCV-endemic regions of the world.

      Acknowledgements

      This work was facilitated by a Fellowship Appointment to the Research Participation Program administered by the Oak Ridge Institute for Science and Education (ORISE) at the Center for Biologics Evaluation and Research (CBER) through an interagency agreement between the US Department of Energy (DOE) and the US Food and Drug Administration (FDA) . The design, execution, findings, and conclusions of this study are those of the authors and should not be construed as representative of ORISE, FDA, DOE, the Department of Health and Human Services, or the Morgan State University.
      Conflict of interest: Dougbeh-Chris Nyan, MD and Kevin L. Swinson, MSc have patent applications pending.
      Authors’ contributions: Dougbeh Chris Nyan conceived the study, designed and conducted the experiments, and drafted the manuscript for publication. Kevin L. Swinson contributed to conception, experimental design, data analysis, and reviewed the manuscript. All authors approved the manuscript for publication.

      Appendix A. Supplementary data

      Figure S1 Detection of HCV genotypes.
      Figure S2 Identification of HCV genotypes in clinical specimens.
      Figure S3 Detection of HCV-RNA with heat-treated plasma specimens.
      Table S1 Evaluation of HCV RT-LAMP genotyping assay with clinical donor specimens.

      References

        • Moratorio G.
        • Martínez M.
        • Gutiérrez M.F.
        • González K.
        • Colina R.
        • López-Tort F.
        • et al.
        Evolution of naturally occurring 5′ non-coding region variants of hepatitis C virus in human populations of the South American region.
        Virol J. 2007; 4: 79
        • Ghany M.G.
        • Strader D.B.
        • Thomas D.L.
        • Seeff L.B.
        Diagnosis, management, and treatment of hepatitis C: an update.
        Hepatology. 2009; 4: 1335-1374
        • Liang T.J.
        • Rehermann B.
        • Seef L.B.
        • Hoofnagel J.H.
        Pathogenesis, Natural History, Treatment, and Prevention of Hepatitis C.
        Ann Int Med. 2000; 132: 296-305
      1. NIH consensus statement on management of hepatitis C: 2002.
        NIH Consens State Sci Statements. 2002; 19: 1-46
        • Messina J.P.
        • Humphreys I.
        • Flaxman A.
        • Brown A.
        • Cooke G.S.
        • Pybus O.G.
        • et al.
        Global distribution and prevalence of hepatitis C virus genotypes.
        Hepatology. 2015; 61: 77-87https://doi.org/10.1002/hep.27259
        • Gower E.
        • Estes C.
        • Blach S.
        • Razavi-Shearer K.
        • Razavi H.
        Global epidemiology and genotype distribution of the hepatitis C virus infection.
        J Hepatol. 2014; 61: S45-S57https://doi.org/10.1016/j.jhep.2014.07.027
        • Zein N.
        Clinical significance of hepatitis C virus genotype.
        Clin Microbiol Rev. 2000; 13: 223-235
        • Smith D.B.
        • Bukh J.
        • Kuiken C.
        • Muerhoff A.S.
        • Rice C.M.
        • Stapleton J.T.
        • et al.
        Expanded classification of hepatitis C virus into 7 genotypes and 67 subtypes: united criteria and genotype assignment Web resource.
        Hepatology. 2014; 59: 318-327
        • Lamballerie X.
        • Charrel R.N.
        • Attoui A.H.
        • De Micco P.
        Classification of hepatitis C virus variants in six major types based on analysis of the envelope 1 and nonstructural 5B genome regions and complete polyprotein sequences.
        J Gen Virol. 1997; 78: 45-51
        • Simmonds P.
        • Alberti A.
        • Alter H.J.
        • Bonino F.
        • Bradley D.W.
        • Brechot C.
        • et al.
        A proposed system for the nomenclature of hepatitis C viral genotypes.
        Hepatology. 1994; 19: 1321-1324
      2. Infectious Diseases Society of America (IDSA). Recommendations for testing, managing and treatment of hepatitis C. IDSA; 2014. Available at: www.hcvguidelines.org (Accessed date: November 3, 2015).

        • De Leuw P.
        • Sarrazin C.
        • Zeuzem S.
        How to use virological tools for the optimal management of chronic hepatitis C.
        Liver Int. 2011; 31: 3-12
        • Etoh R.
        • Imazeki F.
        • Kurihara T.
        • Fukai K.
        • Fujiwara K.
        • Arai M.
        • et al.
        Pegylated interferon-alfa-2a monotherapy in patients infected with HCV genotype 2 and importance of rapid virological response.
        BMC Res Notes. 2011; 24: 316
        • Rho J.
        • Ryu J.S.
        • Hur W.
        • Kim C.W.
        • Jang J.W.
        • Bae S.H.
        • et al.
        Hepatitis C virus (HCV) genotyping by annealing reverse transcription-PCR products with genotype-specific capture probes.
        J Microbiol. 2008; 46: 81-87
        • Nolte F.S.
        • Thurmond C.
        • Fried F.W.
        Preclinical evaluation of AMPLICOR hepatitis C virus test for detection of hepatitis C virus RNA.
        J Clin Microbiol. 1995; 33: 1775-1778
        • Sábato M.F.
        • Shiffman M.L.
        • Langley M.R.
        • Wilkinson D.S.
        • Ferreira-Gonzalez A.
        Comparison of performance characteristics of three real-time reverse transcription-PCR test systems for detection and quantification of hepatitis C virus.
        J Clin Microbiol. 2007; 45: 2529-2536
        • Duarte C.A.
        • Foti L.
        • Nakatani S.M.
        • Riediger I.N.
        • Poersch C.O.
        • Pavoni D.P.
        • et al.
        A novel hepatitis C virus genotyping method based on liquid microarray.
        PLoS One. 2010; 5 (pii: e12822. http://dx.doi.org/10.1371/journal.pone.0012822)
        • De Keukeleire S.
        • Descheemaeker P.
        • Reynders M.
        Diagnosis of hepatitis C virus genotype 2k/1b needs NS5B sequencing.
        Int J Infect Dis. 2015; 41: 1-2https://doi.org/10.1016/j.ijid.2015.10.010
        • Notomi T.
        • Okayama H.
        • Masubuchi H.
        • Yonekawa T.
        • Watanabe K.
        • Amino N.
        • et al.
        Loop-mediated isothermal amplification of DNA.
        Nucleic Acids Res. 2000; 28: E63
        • Nagamine K.
        • Hasse T.
        • Notomi T.
        Accelerated reaction by loop-mediated isothermal amplification using loop primers.
        Mol Cell Probes. 2002; 16: 223-229
        • Blomström A.L.
        • Hakhverdyan M.
        • Reid S.M.
        • Dukes J.P.
        • King D.P.
        • Belák S.
        • et al.
        A one-step reverse transcriptase loop-mediated isothermal amplification assay for simple and rapid detection of swine vesicular disease virus.
        J Virol Methods. 2008; 147: 188-193
        • Kargar M.
        • Askari A.
        • Doosti A.
        • Ghorbani-Dalini S.
        Loop-mediated isothermal amplification assay for rapid detection of hepatitis C virus.
        Indian J Virol. 2012; 23: 18-23
        • Yang J.
        • Fang M.X.
        • Li J.
        • Lou G.Q.
        • Lu H.J.
        • Wu N.P.
        Detection of hepatitis C virus by an improved loop-mediated isothermal amplification assay.
        Arch Virol. 2011; 156: 1387-1396
        • Wang Q.Q.
        • Zhang J.
        • Hu J.S.
        • Chen H.T.
        • Du L.
        • Wu L.Q.
        • et al.
        Rapid detection of hepatitis C virus RNA by a reverse transcription loop-mediated isothermal amplification assay.
        FEMS Immunol Med Microbiol. 2011; 8: 144-147
        • Sun B.
        • Rodriguez-Manzano J.
        • Selck D.A.
        • Khorosheva E.
        • Karymov M.A.
        • Ismagilov R.F.
        Measuring fate and rate of single-molecule competition of amplification and restriction digestion, and its use for rapid genotyping tested with hepatitis C viral RNA.
        Angew Chem Int Ed Engl. 2014; 53: 8088-8092https://doi.org/10.1002/anie.201403035
        • Bukh J.
        • Purcell R.H.
        • Miller R.H.
        Sequence analysis of the 5′ noncoding region of hepatitis C virus.
        Proc Natl Acad Sci U S A. 1992; 89: 4942-4946
        • Ginocchio C.C.
        Life beyond PCR: alternative target amplification technologies for the diagnosis of infectious diseases, part II.
        Clin Microbiol Newsl. 2004; 26: 129-136
        • Nyan D.C.
        • Ulitzky L.E.
        • Cehan N.
        • Williamson P.
        • Winkelman V.
        • Rios M.
        • et al.
        Rapid detection of hepatitis B virus in blood plasma by a specific and sensitive loop-mediated isothermal amplification assay.
        Clin Infect Dis. 2014; 59: 16-23
        • Albertoni G.
        • Arnoni C.P.
        • Araújo P.R.
        • Carvalho F.O.
        • Barreto J.A.
        Signal to cut-off (S/CO) ratio and detection of HCV genotype 1 by real-time PCR one-step method: is there any direct relationship?.
        Braz J Infect Dis. 2010; 14: 147-152
        • Detmer J.
        • Arnoni C.P.
        • Araújo P.R.
        • Carvalho F.O.
        • Barreto J.A.
        Accurate quantification of hepatitis C virus (HCV) RNA from all HCV genotypes by using branched-DNA technology.
        J Clin Microbiol. 1996; 34: 901-907
        • Kotwal G.J.
        • Baroudy B.M.
        • Kuramoto I.K.
        • McDonald F.F.
        • Schiff G.M.
        • Holland P.V.
        • et al.
        Detection of acute hepatitis C virus infection by ELISA using a synthetic peptide comprising a structural epitope.
        Proc Natl Acad Sci U S A. 1992; 89: 4486-4489
        • Hara K.
        • Rivera M.M.
        • Koh C.
        • Sakiani S.
        • Hoofnagle J.H.
        • Heller T.
        Important factors in reliable determination of hepatitis C virus genotype by use of the 5′ untranslated region.
        J Clin Microbiol. 2013; 51: 1485-1489
        • Shemis M.A.
        • El-Abd D.M.
        • Ramadan D.I.
        • El-Sayed M.I.
        • Guirgis B.S.
        • Saber M.A.
        • et al.
        Evaluation of nested polymerase chain reaction for routine hepatitis virus genotyping in Egyptian patients.
        Hepat Mon. 2012; 12: 265-270
        • Nkontchou G.
        • Ziol M.
        • Aout M.
        • Lhabadie M.
        • Baazia Y.
        • Mahmoudi A.
        • et al.
        HCV genotype 3 is associated with a higher hepatocellular carcinoma incidence in patients with ongoing viral C cirrhosis.
        J Viral Hepat. 2011; 18: e516-e522
        • Bruno S.
        • Silini E.
        • Crosignani A.
        • Borzio F.
        • Leandro G.
        Hepatitis C virus genotypes and risk of hepatocellular carcinoma in cirrhosis: a prospective study.
        Hepatology. 1997; 25: 754-758
        • El-Shamy A.
        • Hotta H.
        Impact of hepatitis C virus heterogeneity on interferon sensitivity: an overview.
        World J Gastroenterol. 2014; 20: 7555-7569
        • Sherman K.E.
        • Flamm S.L.
        • Afdhal N.H.
        • Nelson D.R.
        • Sulkowski M.S.
        • Everson G.T.
        • et al.
        Response-guided telaprevir combination treatment for hepatitis C virus infection.
        N Engl J Med. 2011; 365: 1014-1024
        • Maekawa S.
        • Enomoto N.
        Viral factors influencing the response to the combination therapy of peginterferon plus ribavirin in chronic hepatitis C.
        J Gastroenterol. 2009; 44: 1009-1015