Diagnosis of Hepatitis C Virus Infection by Enzyme-linked
Immunosorbent Assay and Reverse Transcriptase-Nested
Polymerase Chain Reaction: A Comparative Evaluation
Menha Swellam1, Magda Sayed Mahmoud1and Adel Abdel-Fatah Ali2
1Department of Biochemistry, Genetic Engineering and Biotechnology Research Division,
National Research Center, Dokki, Giza, Egypt
2Department of Internal Medicine, Faculty of Medicine, Alazhar University, Cairo, Egypt
Hepatitis C virus is one of the main causes of chronic hepati-
tis in developing countries. The current study was to evaluate
the efficacy of the enzyme-linked immunosorbent assay third
generation (ELISA-3) for detection of antibodies to hepatitis C
virus (anti-HCV) in comparison with reverse transcriptase-
nested polymerase chain reaction (RT-nested PCR) to detect
HCV RNA for the diagnosis of hepatitis C virus. Serum sam-
ples were collected from 151 chronic hepatitis C patients and
50 healthy individuals. All samples were tested for anti-HCV
antibodies using ELISA-3 and HCV RNA by RT-nested PCR.
Of the 151,120 (78.9%) were found to be seropositive by
ELISA-3, and 148 (98%) patients were HCV RNA positive, 118
(78.1%) were positives for both, 30 (19.9%) were positive for
ELISA-3 and negative for RT-PCR, and 2 cases (1.3%) were
positive for RT-nested PCR and negative for ELISA-3. The sen-
sitivity and the specificity for the detection of HCV were abso-
lute when the two techniques were combined. In conclusion,
ELISA-3 is a suitable assay for routine screening for anti-HCV.
RT-nested PCR for HCV is a value for the early detection of
viremic, anti-HCV negative cases; this may be of importance in
treatment of hepatitis C.
? 2011 IUBMB
IUBMB Life, 63(6): 430–434, 2011
hepatitic C virus; ELISA; RT-nested PCR.
Hepatitis C virus (HCV) is recognized as a major threat to
global public health (1). The recently published Egyptian De-
mographic Health Survey in 2009 was a national probability
sample of the resident Egyptian population (2). This report esti-
mated an overall anti-HCV antibody prevalence of 18.5%,
reaching 45% in males over 40 years and 30% in females over
50 years (3). The number of Egyptians estimated to be chroni-
cally infected was 9.8%, and approximately, 20% of Egyptian
blood donors are anti-HCV positive, and the strong homogene-
ity of HCV subtypes found in Egypt (mostly 4a) suggests an
epidemic spread of HCV (4). This report provides a precise
national prevalence estimate and includes additional data on
patterns of HCV prevalence by gender, age, urban versus rural,
and between different regions of the country (5).
Laboratory assays that are available for the diagnosis and
management of HCV infection include: (a) serological tests to
detect HCV antibodies [enzyme-linked immunosorbent assay
(ELISA)], (b) molecular tests to detect and quantitate HCV
RNA, and (c) genotyping (6, 7). Serological assays for detecting
anti-HCV antibody cannot distinguish between patients with
active infection and those who have cleared the virus, and due
to the absence of an efficient in vitro culture system for HCV
or assays capable of detecting viral antigens, direct detection of
HCV has depended on nucleic acid amplification technology
(NAT) techniques (8). NAT tests are based on nucleic acid
amplification (PCR and transcription-mediated amplification)
and are currently used to detect viremia (9).
The purpose of this study was to compare the efficacy of
ELISA-3 and reverse transcriptase-nested polymerase chain
reaction (RT-nested PCR) for the diagnosis of hepatitis C virus.
MATERIALS AND METHODS
To achieve our goal in this prospective study, a total of 151
patients with chronic hepatitis C were recruited from the Inter-
Address correspondence to: Prof. Dr. Menha Swellam, Department
of Biochemistry, Genetic Engineering and Biotechnology Research Di-
vision, National Research Center, Tahrir Street, Dokki, Giza 12622,
Egypt. Tel: 12023335451. Fax: 12023370931. E-mail: menha_m_
email@example.com or firstname.lastname@example.org
Received 4 February 2011; accepted 7 March 2011
ISSN 1521-6543 print/ISSN 1521-6551 online
IUBMBLife, 63(6): 430–434, June 2011
nal Medicine Division, Kobry El-Koba Military Hospital. Diag-
nosis of those patients was based on clinical examination, liver
enzymes, detectable anti-HCV antibodies in their sera (Axium
HCV Rapid test, FL), and semiquantitative RT-PCR. None of
the patients was on antiviral treatment. A total of 50 sera were
collected from healthy volunteers having no history of any liver
complications, undetectable anti-HCV antibodies, and negative
HCV RNA by RT-nested PCR in their sera were included as
All samples were separated into three aliquots: the first for
serological tests, the second for nested PCR, and the third re-
served for any necessary result confirmation. The sera were
stored at 280 8C.
for all serum samples using their available commercial kits
according to the manufacturer’s instructions (BioMerieux and
Randox Chem Company, Ghent, Belgium): alanine aminotransfer-
ase (ALT), aspartate aminotransferase (AST) (10), total bilirubin
(T.BIL) (11), alkaline phospatase (ALP) (12), gamma-glutamyl
transpeptidase (GGT) (13), total proteins (14), and albumin (15).
Biochemical laboratory assays were carried out
Enzyme-linked Immunosorbent Assay.
detected by a third generation HCV ELISA [ELISA HCV 3.0
system (Ortho-Clinical Diagnostics, Test System Enhanced
Save)). The ELISA HCV 3.0 uses microwells coated with a
combination of recombinant HCV-encoded antigens (c22-3,
c200, and NS5) originating from four regions of the viral ge-
nome (core, NS3, NS4, and NS5). The assay was performed
using the manufacturer’s procedures based on ref. 16.
Anti-HCV antibody was
Reverse Transcriptase Nested Polymerase Chain Reaction.
a simple and rapid method described in ref. 17, authors have
used reverse transcriptase nested polymerase chain reaction
(RT-nested PCR) to amplify a fragment of the 50untranslated
region (50UTR) of the HCV RNA genome that does not require
the RNA extraction step. In brief, after denaturation (30 sec at
92 8C), 3 ll of serum was added to the RT mixture (50 mM
Tris–HCl, pH 8.2, 70 mM KCl, 10 mM MgCl2, 4 mM DTT, 12
U of human placental ribonuclease inhibitor (RNA guard, Phar-
macia, Uppsala, Sweden), 0.4% Nonidet P-40, 50 pmol of a
specific antisense primer (R1), 250 mM dNTPs, and 6 U of
avian myeloblastosis virus reverse transcriptase (AMV-RT,
United State Biochemical, Cleveland, OH), in a final volume of
25 ll). RT was carried out at 42 8C for 60 min and the result-
ing cDNA was immediately denatured by heating for 5 min at
100 8C. Amplification was performed following the nested pri-
mers protocol (18), as previously described (19) with two sets
of primers located in the 50untranslated region (UTR) of the
viral genome. The first round of PCR (35 cycles) was carried
out using primers A1 (50-GATGCACGGTCTACGAGACCTC-
30) and S1 (50-AACTACTGTCTTCACGCAGAA-30) generating
a PCR product of 289 bp. For the second round (25 cycles), we
used primers A2 (50-GCGACCCAACACTACTCGGCT-30) and
S2 (50-ATGGCGTFAGTATGAGTG-30) generating a PCR prod-
uct of 187 bp. PCR cycles were as follows: denaturation at
94 8C for 1 min, annealing of primers at 45 8C for 1 min, and
elongation at 72 8C for 2 min in a DNA Thermal Cycler
(Perkin Elmer Cetus, Foster City, CA). The final amplification
cDNA product was mixed with bromophenol blue, electropho-
resed in a 2% agarose gel, stained with ethidium bromide,
visualized, and photographed (Fig. 1).
SPSS version 10.0 for windows was used for the analysis of
data and summary statistics. The results for all variables were
set in the form of rates (%). Fisher’s exact and v2tests were
applied to find out the association among the categorical varia-
bles. Sensitivity (number of positive cases of hepatitis C virus)
and specificity (number of negative cases of healthy volunteers)
were calculated according to standard statistical methods (20).
Linear regression analysis was carried out to examine the rela-
tion between the applied techniques and the routine biochemical
markers. P value less than 0.05 was considered as significant.
Among the 151 samples collected from chronic hepatitis
patients, 113 were males and the remaining (n 5 38) were
Figure 1. Agarose gel electrophoresis patterns of reverse tran-
scriptase-nested polymerase chain reaction (RT-nested PCR)
products of hepatitis C patients. Lane 1: DNA size marker 100
bp; lanes 2, 4, and 5: RT-nested PCR products of HCV positive
(289 bp); and lanes 3 and 6: RT-nested PCR products of HCV
431DIAGNOSIS OF HEPATITIS C VIRUS BY ELISA AND RT-NESTED PCR
females with matched age (mean age, 40 6 10 years; range,
35–41 years). Also 50 blood samples were collected from
healthy volunteers (35 males and 15 females) with matched age
(mean age, 38 6 11 years; range, 33–41 years). Demographic
characteristics and routine examinations are reported in Table 1.
Biochemical laboratory tests were significantly higher in chronic
hepatitis C patients when compared with the healthy volunteers.
Frequency of Samples Detected by ELISA-3
and RT-nested PCR
As shown in Table 2, significant difference was detected
between the study population and the two techniques of the
study. Of 151 chronic hepatitis C patients, 78.9% and 98%
revealed positive using ELISA-3 and RT-nested PCR, respec-
Comparison Between ELISA-3 and RT-nested PCR
When authors compared between ELISA-3 and RT-nested
PCR for the detection of chronic HCV, true positive cases were
78.1% (118/151) and true negative individuals were absolute, as
shown in Table 3. Thirty (19.9%) HCV patients were positive
using RT-nested PCR, however, their ELISA-3 results were
Evaluation of Validity and Diagnostic Power of ELISA-3
and RT-nested PCR for Detecting HCV
The specificity of ELISA-3 and RT-nested PCR for the
detection of HCV was absolute for both, as shown in Table 4.
The diagnostic index of ELISA-3 (178.9%) was lower than that
of the RT-nested PCR (198%). Also the agreement rate of
ELISA-3 was lower than that of RT-nested PCR (Table 4).
Correlation Between ELISA-3 and RT-nested PCR with
Routine Biochemical Markers
Significant direct correlations were reported between ALT,
AST, ALP, GGT, and T.BIL with ELISA-3 (R 5 0.912, 0.828,
0.813, 0.317, and 0.813, respectively, at P \ 0.001) and RT-
PCR (R 5 0.719, 0.632, 0.579, 0.29, and 0.6, respectively, at P
\ 0.001). On the other hand, reverse correlations were detected
between them with total protein and albumin (R 5 20.305,
20.576 for ELISA-3 and 20.22 and 20.398 for RT-PCR,
respectively, at P \ 0.001). The correlation between the two
investigated techniques revealed a direct correlation (R 5
0.682, P \ 0.001).
Since the identification and molecular characterization of
hepatitis C virus in 1989 (21), a variety of diagnostic tests
based on the detection of anti-HCV antibodies in serum samples
have been developed and refined. Among them, recombinant
immunoblot assay (RIBA), which identifies antibodies to indi-
vidual HCV antigens (22), and also three generations of sero-
diagnostic anti-HCV antigen tests have been developed, with
each new generation providing incremental improvements in the
sensitivity to anti-HCV antibodies. The third-generation ELISAs
Demographic characteristics and routine examinations (mean 6 SE) for the study population
Healthy volunteers (n 5 50) Hepatitis C patients (n 5 151) Statistics F, P value
Mean age (range) years
Male (n 5 148)
Female (n 5 53)
38 6 11 (33–41)40 6 10 (35–41)2.4, 0.4
23.7 6 0.3
27.8 6 0.3
0.5 6 0.005
158.9 6 3.3
24.8 6 0.3
11.26 6 2.2
4.3 6 0.04
78.5 6 0.8
62.22 6 0.83
0.85 6 0.008
196.6 6 4.6
67.7 6 1.2
5.2 6 0.05
3.5 6 0.54
Frequency of samples detected by both
ELISA-3 and RT-nested PCR
(n 5 50)
(n 5 151)
Negative (n 5 53)
Positive (n 5 148)
Negative (n 5 81)
Positive (n 5 120)
432 SWELLAM ET AL.
are designed to detect antibodies to four recombinant HCV pro-
teins, and it was reported to be more specific than their prede-
cessors (23). Recently, a variety of home-brew or in-house PCR
assays to test for the seropresence of HCV RNA are available.
Many researches considered that direct molecular qualitative
detection of HCV RNA by reverse transcription (RT) and PCR
is the gold standard for the diagnosis of HCV infection (1, 4).
The current study was carried out to compare between the two
techniques (i.e., ELISA-3 and RT-nested PCR) for the better di-
agnosis of HCV infection.
The results of both ELISA-3 and RT-nested PCR revealed
significant difference between control individuals and HCV
patients. These findings indicate that both are sufficient for the
diagnosis of HCV infection in clinical laboratories. In this
study, a number of false negative cases detected using RT-
nested PCR were smaller (3, 2%) than those detected by
ELISA-3 (31, 20.5%). Indicating that PCR-based assays are
able to ascertain minute amounts of HCV RNA in serum as pre-
viously reported (6) that PCR helps to resolve weakly positive
or negative ELISA results when clinical signs and/or risk fac-
tors are compatible with HCV infection. Also, ELISA is a
method to detect antibody for the purpose of diagnosis of virus
infection. The antibody was only detected 1–2 weeks after
infection, which reflected the immune response of the host, but
could not explain the virus replication. PCR method could
directly detect the virus nucleic acids. It could reflect the state
of virus replication. When the virus was cleaned up, only the
antibody was positive, the nucleic acids could not be detected.
Hence, the detection rate of PCR was lower when ELISA was
used as a golden standard (24).
The differentiation between acute and chronic HCV infection
depends on the clinical presentation. After acute exposure,
detection of HCV RNA usually precedes the detection of anti-
body reactivity in serum; HCV RNA can be identified as early
as 2 weeks following exposure, whereas anti-HCV antibodies
are generally not detected before 8–12 weeks (25) and hence,
the positive response between these two techniques requires
careful analysis for interpretation. In the present study, 78.9%
of HCV patients were both RT-nested PCR and ELISA-3 posi-
tive indicating that the HCV infection is acute or chronic
depending on the clinical context. In 19.9% of HCV samples,
results were positive by RT-nested PCR and negative by
ELISA-3; this might indicate the early acute HCV infection,
chronic HCV in chronic immunosuppressed patients or false-
positive HCV test. Negative RT-nested PCR results and positive
ELISA-3 were reported in two HCV cases (1.3%) which might
indicate the resolution of HCV, acute HCV during the period of
low-viremia, or false anti-HCV positive. Only one case was
reported negative for both and could be due to the absence of
anti-HCV antibodies or any contamination in RT-nested PCR
Our results revealed significant direct correlation between
ELISA-3 and RT-nested PCR with routine biochemical tests. In
chronic hepatitis C, increases in the alanine and asparate amino-
transferases range from 1 to 20 times (but usually less than five
times) the upper limit of normal. Alanine aminotransferase
(ALT) levels were higher than aspartate aminotransferase (AST)
levels but that finding was reversed in patients, who have
cirrhosis. Alkaline phosphatase and gamma-glutamyl transpepti-
dase were normal in 10 cases and elevated in the remaining,
which might indicate cirrhosis (9).
The sensitivity and the specificity of ELISA-3 in the current
study were 78.9% and 100%, respectively; these are good
enough for a diagnostic assay. Similarly, the specificity of RT-
PCR was absolute at high sensitivity (98%) indicating that it is
not only suitable for clinical diagnosis but also suitable for the
Comparison between ELISA-3 and RT-nested PCR results
(%) (n 5 201)
(n 5 50)
(n 5 151)
Positive ELISA-3 or positive RT-nested PCR
Negative ELISA-3 or negative RT-nested PCR
Negative ELISA-3 or positive RT-nested PCR
Positive ELISA-3 or negative RT-nested PCR
X25 93.58; P \ 0.0001
X25 195; P \ 0.0001
Diagnostic power of ELISA-3 and RT-nested PCR
Sensitivity SpecificityPPV NPV Agreement rate Diagnostic index
ELISA-3 1 RT-nested PCR
433DIAGNOSIS OF HEPATITIS C VIRUS BY ELISA AND RT-NESTED PCR
screening of HCV to prevent the transmission of this disease.
Interestingly, when authors combined both techniques as shown
in Table 4, the sensitivity and the specificity were absolute and
the diagnostic index was 200% indicating that it is advisable to
confirm reactive samples using the two methods, and their com-
bination can be useful in epidemiological studies. In these
respects, a study in progress is carried out in a large-scale popu-
In conclusion, ELISA assays have many advantages in the
diagnostic setting including ease of automation, ease of use,
relative cost-effectiveness, and low variability. However, as
with all enzyme immunoassays, false-positive results are occa-
sionally a problem with a ELISA-3, additional or confirmatory
testing is often helpful like RT-nested PCR. Moreover, further
studies are recommended to study hepatitis C genotypes to
facilitate improved clinical outcomes and epidemiologic studies
and to provide information that has major implications for clini-
cal management of hepatitis C and possible HCV vaccine de-
1. Ayesh, B. M., Zourob, S. S., Abu-Jadallah, S. Y., and Shemer-Avni, Y.
(2009) Most common genotypes and risk factors for HCV in Gaza strip:
a cross sectional study. Virol. J. 6, 105–112.
2. El-Zanaty, F. and Way, A. (2009) Egypt Demographic and Health Sur-
vey 2008. Ministry of Health (El-Zanaty and Associates and Macro
International), Cairo, Egypt, pp. 431.
3. Mohamed, M. K., Bakr, I., El-Hoseiny, M., Arafa, N., Hassan, A.,
Ismail, S., Anwar, M., Attala, M., Rekacewicz, C., Zalata, K., Abdel-
Hamid, M., Esmat, G., and Fontanet, A. (2006) HCV-related morbidity
in a rural community of Egypt. J. Med. Virol. 78, 1185–1189.
4. World Health Organization (2002) Hepatitis C. WHO, Geneva.
5. Miller, F. D. and Abu-Raddad, L. J. (2010) Evidence of intense ongoing
endemic transmission of hepatitis C virus in Egypt. Proc. Natl. Acad.
Sci. USA 107, 14757–14762.
6. Tashkandy, M. A., Khodari, Y. A., Ibrahim, A. M., Dhafar, K. O., Gaz-
zaz, Z. J., and Azab, B. A. (2007) Evaluation of the available anti-HCV
antibody detection tests and RT-PCR assay in the diagnosis of hepatitis
C virus infection. Saudi J. Kidney Dis. Transpl. 18, 523–531.
7. Ali, A., Ahmed, H., and Idrees, M. (2010) Molecular epidemiology of
Hepatitis C virus genotypes in Khyber Pakhtoonkhaw of Pakistan. Virol.
J. 7, 203–210.
8. Fabrizi, F., Lunghi, G., Aucella, F., Mangano, S., Barbisoni, F., Bisegna,
S., Vigilante, D., Limido, A., and Martin, P. (2005) Novel assay using
total hepatitis C virus (HCV) core antigen quantification for diagnosis of
HCV infection in dialysis patients. J. Clin. Microbiol. 43, 414–420.
9. Berry, V., Arora, R., and Paul, P. (2005) Hepatitis C-clinical outcome
and diagnosis. JK Sci. 7, 129–135.
10. Wikison, J. H., Baron, D. N., Moss, D. W., and Walker, P. G. (1972)
Standardization of clinical enzyme assays: a reference method for asparate
and alanine transaminases. J. Clin. Pathol., 25, 940–945.
11. Parviainen, M. T. (1997) A modification of the acid diazo coupling
method (Malloy-Evellyn) for the determination of serum total bilirubin.
Scan. J. Clin. Lab. Invest. 57, 275–279.
12. Roy, A. V. (1970) Rapid method for determining alkaline phosphatase activ-
ity in serum with thymolphthalein monophosphate. Clin. Chem. 16, 431–436.
13. Persijn, J. P. and Van der Silk, W. (1976) A new method for the deter-
mination of gamma-glutathione in serum. J. Clin. Chem. 14, 421–427.
14. Peters, T. (1968) Proposals for standardization of total protein assays.
Clin. Chem. 14, 1147–1150.
15. Doumas, B. (1972) Colorimetric method for determination of plasma
albumin. Clin. Chim. Acta 18, 212–215.
16. Esteban, J. I., Gonzalea, A., Hernandez, J. M., Viladomiu, L., Sanchez,
C., Vidal, X., Esetaban, R., and Guradia, J. (1991) Evaluation of anti-
bodies to hepatitis C virus a study of transfusion-associated hepatitis.
N. Eng. J. Med. 323, 1107–1112.
17. Ravaggi, A., Primi, D., and Cariani, E. (1992) Direct PCR amplification
of HCV RNA from human serum. Genome Res. 1, 291–292.
18. Garson, J. A., Tedder, R. S., Briggs, M., Tuke, P., Glazebrook, J. A.,
Trute, A., Parker, D., Barbara, Contreras, M., and Aloysius, S. (1990).
Detection of hepatitis C viral sequences in blood donations by ‘‘nested’’
polymerase chain reaction and prediction of infectivity. Lancet 335,
19. Imberti, L., Cariani, E., Bettinardi, A., Zonaro, A., Albertini, A., and
Primi, D. (1991). An immunoassay for specific amplified HCV sequen-
ces. J. Virol. Methods 34, 233–243.
20. Sackett, D. L., Haynes, R. B., Guyatt, G. H., and Tugwell, P. (1991)
Clinical Epidemiology, 2nd ed. Little Brown, Boston.
21. Kuo, G., Choo, Q. L., and Aher, H. J. (1989) An assay for circulating
antibodies to a major etiologic virus of non-A non-B viral hepatitis. Sci-
ence 244, 362–364.
22. Gadano, A., Galdame, O., and Marciano, S. (2010) Diagnosis of patients
with suspected chronic hepatitis C infection. Ann. Hepatol. 9, S34–S38.
23. Wu, F.-B., Ouyan, H.-Q., Tang, X.-Y., and Zhou, Z.-X. (2008) Double-
antigen sandwich time-resolved immunofluorometric assay for the detec-
tion of anti-hepatitis C virus total antibodies with improved specificity
and sensitivity. J. Med. Microb. 57, 947–953.
24. Wang, N., Gao, X.-Q., and Han, J.-X. (2004) Simultaneous detection of
HBV and HCV by multiplex PCR normalization. World J. Gastroen-
terol. 10, 2439–2443.
25. Ghany, M. G., Strader, D. B., Thomas, D. L., and Steef, L. B. (2009)
Diagnosis, management of treatment of hepatitis C anuodate. Hepato-
logy 49, 1335–1374.
434SWELLAM ET AL.