Skip to main content

A rapid, low-cost quantitative diagnostic method for hepatitis C virus infection using capillary zone electrophoresis

Abstract

Hepatitis C virus (HCV)-RNA amplification is a costly procedure in terms of time and reagents. Consequently, the search for more a cost-effective specific HCV diagnostic method is of great interest. Capillary zone electrophoresis (CZE) methods that detect HCV in serum, plasma, whole blood, and ascites without the need for sample pretreatment are not currently available. Here, a CZE method was developed that detects a larger specific peak in serum and other body fluids of HCV-infected patients than that found in healthy or hepatitis B virus (HBV)-infected individuals. The nature of the HCV peak was investigated using biochemical treatments, including RNase, DNase, and chymotrypsin enzymes. Electroeluted HCV peak was applied to transmission electron microscopy; electron micrographs showed that the HCV peak was attributed to virus-like particles with diameter and morphological properties similar to non-enveloped HCV nucleocapsids. The determination of CZE-HCV and HCV-RNA levels using quantitative real-time reverse transcriptase-polymerase chain reaction (qRT-PCR) in 258 subjects revealed that these two tests were highly correlated (r = 0.92, p < 0.0001). One important issue of HCV testing is the storage conditions of serum to obtain reliable results. Serum samples at −20 °C showed the best preservation of the HCV peak up to one year. In conclusion, we detected HCV using CZE in a microliters volume from different body fluids. Besides the stability of samples in maintaining their peak height, the HCV-CZE test is rapid (<15 min) and a well-suited and low-cost technique. Thus, a major improvement in the quantitative diagnosis of HCV infection was established.

This is a preview of subscription content, access via your institution.

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7

References

  1. 1.

    Sainz B Jr, Barretto N, Martin DN, Hiraga N, Imamura M, Hussain S, Marsh KA, Yu X, Chayama K, Alrefai WA, Uprichard SL (2012) Identification of the Niemann–Pick C1-like 1 cholesterol absorption receptor as a new hepatitis C virus entry factor. Nat Med 18:281–285

    CAS  PubMed Central  PubMed  Article  Google Scholar 

  2. 2.

    Lanford RE, Hildebrandt-Eriksen ES, Petri A, Persson R, Lindow M, Munk ME, Kauppinen S, Ørum H (2010) Therapeutic silencing of microRNA-122 in primates with chronic hepatitis C virus infection. Science 327:198–201

    CAS  PubMed Central  PubMed  Article  Google Scholar 

  3. 3.

    Shepard CW, Finelli L, Alter MJ (2005) Global epidemiology of hepatitis C virus infection. Lancet Infect Dis 5:558–567

    PubMed  Article  Google Scholar 

  4. 4.

    Park Y, Lee JH, Kim BS, Kim do Y, Han KH, Kim HS (2010) New automated hepatitis C virus (HCV) core antigen assay as an alternative to real-time PCR for HCV RNA quantification. J Clin Microbiol 48:2253–2256

    PubMed Central  PubMed  Article  Google Scholar 

  5. 5.

    Lerat H, Hollinger FB (2004) Hepatitis C virus (HCV) occult infection or occult HCV RNA detection? J Infect Dis 189:3–6

    PubMed  Article  Google Scholar 

  6. 6.

    Descamps V, Op de Beeck A, Plassart C, Brochot E, François C, Helle F, Adler M, Bourgeois N, Degré D, Duverlie G, Castelain S (2012) Strong correlation between liver and serum levels of hepatitis C virus core antigen and RNA in chronically infected patients. J Clin Microbiol 50:465–468

    CAS  PubMed Central  PubMed  Article  Google Scholar 

  7. 7.

    Bruns T, Steinmetzer K, Ermantraut E, Stallmach A (2009) Hepatitis C virus RNA quantitation in venous and capillary small-volume whole-blood samples. J Clin Microbiol 47:3231–3240

    CAS  PubMed Central  PubMed  Article  Google Scholar 

  8. 8.

    Linhardt RJ, Toida T (2002) Tech.Sight. Capillary electrophoresis. Ultra-high resolution separation comes of age. Science 298:1441–1442

    PubMed  Article  Google Scholar 

  9. 9.

    Desai MJ, Armstrong DW (2003) Separation, identification, and characterization of microorganisms by capillary electrophoresis. Microbiol Mol Biol Rev 67:38–51

    CAS  PubMed Central  PubMed  Article  Google Scholar 

  10. 10.

    Attallah AM, Abdel Malak CA, Elghawalby NA, Shehatta AS, Abdel-Raouf M, Shiha GE (2004) Identification of a specific marker for hepatitis C virus infection using capillary zone electrophoresis. Clin Chim Acta 346:171–179

    CAS  PubMed  Article  Google Scholar 

  11. 11.

    Chang Y, Yang B, Zhao X, Linhardt RJ (2012) Analysis of glycosaminoglycan-derived disaccharides by capillary electrophoresis using laser-induced fluorescence detection. Anal Biochem 427:91–98

    CAS  PubMed Central  PubMed  Article  Google Scholar 

  12. 12.

    Oita I, Halewyck H, Pieters S, Dejaegher B, Thys B, Rombaut B, Heyden YV (2009) Improving the capillary electrophoretic analysis of poliovirus using a Plackett–Burman design. J Pharm Biomed Anal 50:655–663

    CAS  PubMed  Article  Google Scholar 

  13. 13.

    Schnabel U, Groiss F, Blaas D, Kenndler E (1996) Determination of the pI of human rhinovirus serotype 2 by capillary isoelectric focusing. Anal Chem 68:4300–4303

    CAS  PubMed  Article  Google Scholar 

  14. 14.

    Okun VM, Ronacher B, Blaas D, Kenndler E (1999) Analysis of common cold virus (human rhinovirus serotype 2) by capillary zone electrophoresis: the problem of peak identification. Anal Chem 71:2028–2032

    CAS  PubMed  Article  Google Scholar 

  15. 15.

    Mann B, Traina JA, Soderblom C, Murakami PK, Lehmberg E, Lee D, Irving J, Nestaas E, Pungor E (2000) Capillary zone electrophoresis of a recombinant adenovirus. J Chromatogr A 895:329–337

    CAS  PubMed  Article  Google Scholar 

  16. 16.

    Végvári A, Hjertén S (2003) Hybrid microdevice electrophoresis of peptides, proteins, DNA, viruses, and bacteria in various separation media, using UV-detection. Electrophoresis 24:3815–3820

    PubMed  Article  Google Scholar 

  17. 17.

    Mironov GG, Chechik AV, Ozer R, Bell JC, Berezovski MV (2011) Viral quantitative capillary electrophoresis for counting intact viruses. Anal Chem 83:5431–5435

    CAS  PubMed  Article  Google Scholar 

  18. 18.

    Okun VM, Blaas D, Kenndler E (1999) Separation and biospecific identification of subviral particles of human rhinovirus serotype 2 by capillary zone electrophoresis. Anal Chem 71:4480–4485

    CAS  PubMed  Article  Google Scholar 

  19. 19.

    Halewyck H, Oita I, Thys B, Dejaegher B, Vander Heyden Y, Rombaut B (2010) Identification of poliovirions and subviral particles by capillary electrophoresis. Electrophoresis 31:3281–3287

    CAS  PubMed  Article  Google Scholar 

  20. 20.

    Okun VM, Ronacher B, Blaas D, Kenndler E (2000) Affinity capillary electrophoresis for the assessment of complex formation between viruses and monoclonal antibodies. Anal Chem 72:4634–4639

    CAS  PubMed  Article  Google Scholar 

  21. 21.

    Kolesar JM, Allen PG, Doran CM (1997) Direct quantification of HIV-1 RNA by capillary electrophoresis with laser-induced fluorescence. J Chromatogr B Biomed Sci Appl 697:189–194

    CAS  PubMed  Article  Google Scholar 

  22. 22.

    Grassi M, Mammarella A, Sagliaschi G, Granati L, Musca A, Traditi F, Pezzella M (2001) Persistent hepatitis G virus (HGV) infection in chronic hemodialysis patients and non-B, non-C chronic hepatitis. Clin Chem Lab Med 39:956–960

    CAS  PubMed  Article  Google Scholar 

  23. 23.

    Thaitrong N, Liu P, Briese T, Lipkin WI, Chiesl TN, Higa Y, Mathies RA (2010) Integrated capillary electrophoresis microsystem for multiplex analysis of human respiratory viruses. Anal Chem 82:10102–10109

    CAS  PubMed Central  PubMed  Article  Google Scholar 

  24. 24.

    Farías A, Ré V, Mengarelli S, Kremer L, Pisano MB, Allende L, Nicolás J, Elbarcha O, Contigiani M (2010) Detection of hepatitis C virus (HCV) in body fluids from HCV monoinfected and HCV/HIV coinfected patients. Hepatogastroenterology 57:300–304

    PubMed  Google Scholar 

  25. 25.

    Cuhadar S, Atay A, Koseoglu M, Dirican A, Hur A (2012) Stability studies of common biochemical analytes in serum separator tubes with or without gel barrier subjected to various storage conditions. Biochem Med (Zagreb) 22:202–214

    CAS  Article  Google Scholar 

  26. 26.

    Rose DJ, Jorgenson JW (1988) Fraction collector for capillary zone electrophoresis. J Chromatogr 438:23–34

    CAS  PubMed  Article  Google Scholar 

  27. 27.

    Shah VP, Midha KK, Findlay JW, Hill HM, Hulse JD, McGilveray IJ, McKay G, Miller KJ, Patnaik RN, Powell ML, Tonelli A, Viswanathan CT, Yacobi A (2000) Bioanalytical method validation—a revisit with a decade of progress. Pharm Res 17:1551–1557

    CAS  PubMed  Article  Google Scholar 

  28. 28.

    Bland JM, Altman DG (1999) Measuring agreement in method comparison studies. Stat Methods Med Res 8:135–160

    CAS  PubMed  Article  Google Scholar 

  29. 29.

    Petersen JR, Okorodudu AO, Mohammad A, Payne DA (2003) Capillary electrophoresis and its application in the clinical laboratory. Clin Chim Acta 330:1–30

    CAS  PubMed  Article  Google Scholar 

  30. 30.

    Beaubier NT, Hart AP, Bartolo C, Willman CL, Viswanatha DS (2000) Comparison of capillary electrophoresis and polyacrylamide gel electrophoresis for the evaluation of T and B cell clonality by polymerase chain reaction. Diagn Mol Pathol 9:121–131

    CAS  PubMed  Article  Google Scholar 

  31. 31.

    Bergmann J, Jaehde U, Mazereeuw M, Tjaden UR, Schunack W (1996) Potential of on-line isotachophoresis-capillary zone electrophoresis with hydrodynamic counterflow in the analysis of various basic proteins and recombinant human interleukin-3. J Chromatogr A 734:381–389

    CAS  PubMed  Article  Google Scholar 

  32. 32.

    Klein KC, Dellos SR, Lingappa JR (2005) Identification of residues in the hepatitis C virus core protein that are critical for capsid assembly in a cell-free system. J Virol 79:6814–6826

    CAS  PubMed Central  PubMed  Article  Google Scholar 

  33. 33.

    Kunkel M, Watowich SJ (2002) Conformational changes accompanying self-assembly of the hepatitis C virus core protein. Virology 294:239–245

    CAS  PubMed  Article  Google Scholar 

  34. 34.

    Gastaminza P, Dryden KA, Boyd B, Wood MR, Law M, Yeager M, Chisari FV (2010) Ultrastructural and biophysical characterization of hepatitis C virus particles produced in cell culture. J Virol 84:10999–11009

    CAS  PubMed Central  PubMed  Article  Google Scholar 

  35. 35.

    Maillard P, Krawczynski K, Nitkiewicz J, Bronnert C, Sidorkiewicz M, Gounon P, Dubuisson J, Faure G, Crainic R, Budkowska A (2001) Nonenveloped nucleocapsids of hepatitis C virus in the serum of infected patients. J Virol 75:8240–8250

    CAS  PubMed Central  PubMed  Article  Google Scholar 

  36. 36.

    Subirats X, Blaas D, Kenndler E (2011) Recent developments in capillary and chip electrophoresis of bioparticles: viruses, organelles, and cells. Electrophoresis 32:1579–1590

    CAS  PubMed  Google Scholar 

  37. 37.

    Boulant S, Vanbelle C, Ebel C, Penin F, Lavergne JP (2005) Hepatitis C virus core protein is a dimeric alpha-helical protein exhibiting membrane protein features. J Virol 79:11353–11365

    CAS  PubMed Central  PubMed  Article  Google Scholar 

  38. 38.

    Yamashita T, Honda M, Kaneko S (2011) Molecular mechanisms of hepatocarcinogenesis in chronic hepatitis C virus infection. J Gastroenterol Hepatol 26:960–964

    CAS  PubMed  Article  Google Scholar 

  39. 39.

    Quinlan GJ, Martin GS, Evans TW (2005) Albumin: biochemical properties and therapeutic potential. Hepatology 41:1211–1219

    CAS  PubMed  Article  Google Scholar 

  40. 40.

    Wang RE, Tian L, Chang YH (2012) A homogeneous fluorescent sensor for human serum albumin. J Pharm Biomed Anal 63:165–169

    CAS  PubMed Central  PubMed  Article  Google Scholar 

  41. 41.

    Runyon BA; AASLD Practice Guidelines Committee (2009) Management of adult patients with ascites due to cirrhosis: an update. Hepatology 49:2087–2107

    PubMed  Article  Google Scholar 

  42. 42.

    McDade TW, Williams S, Snodgrass JJ (2007) What a drop can do: dried blood spots as a minimally invasive method for integrating biomarkers into population-based research. Demography 44:899–925

    PubMed  Article  Google Scholar 

  43. 43.

    McDade TW, Shell-Duncan B (2002) Whole blood collected on filter paper provides a minimally invasive method for assessing human transferrin receptor level. J Nutr 132:3760–3763

    CAS  PubMed  Google Scholar 

  44. 44.

    Déglon J, Thomas A, Daali Y, Lauer E, Samer C, Desmeules J, Dayer P, Mangin P, Staub C (2011) Automated system for on-line desorption of dried blood spots applied to LC/MS/MS pharmacokinetic study of flurbiprofen and its metabolite. J Pharm Biomed Anal 54:359–367

    PubMed  Article  Google Scholar 

  45. 45.

    Worthman CM, Stallings JF (1997) Hormone measures in finger-prick blood spot samples: new field methods for reproductive endocrinology. Am J Phys Anthropol 104:1–21

    CAS  PubMed  Article  Google Scholar 

  46. 46.

    Sener K, Yapar M, Bedir O, Gül C, Coskun O, Kubar A (2010) Stability of hepatitis C virus RNA in blood samples by TaqMan real-time PCR. J Clin Lab Anal 24:134–138

    CAS  PubMed  Article  Google Scholar 

  47. 47.

    José M, Curtu S, Gajardo R, Jorquera JI (2003) The effect of storage at different temperatures on the stability of hepatitis C virus RNA in plasma samples. Biologicals 31:1–8

    PubMed  Article  Google Scholar 

  48. 48.

    Ray SC, Arthur RR, Carella A, Bukh J, Thomas DL (2000) Genetic epidemiology of hepatitis C virus throughout Egypt. J Infect Dis 182:698–707

    CAS  PubMed  Article  Google Scholar 

Download references

Acknowledgments

The authors would like to thank the staff of the Electron Microscope Unit, Faculty of Science, Ain Shams University, Egypt, for the electron microscopy analysis. This work has been completely supported financially and carried out at the Biotechnology Research Center, New Damietta, Egypt. A. M. Attallah generated the idea and designed the study.

Conflict of interest

All authors had no conflicts of interest.

Author information

Affiliations

Authors

Corresponding author

Correspondence to A. M. Attallah.

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Attallah, A.M., Abdallah, S.O., El-desouky, M.A. et al. A rapid, low-cost quantitative diagnostic method for hepatitis C virus infection using capillary zone electrophoresis. Eur J Clin Microbiol Infect Dis 33, 439–452 (2014). https://doi.org/10.1007/s10096-013-1976-8

Download citation

Keywords

  • Migration Time
  • Ascitic Fluid
  • Capillary Zone Electrophoresis
  • Capillary Zone Electrophoresis Method
  • Capillary Zone Electrophoresis Analysis