Original Article

Detection and Quantification of HCV-RNA by RT-PCR among Anti-HCV Positive Patients

Rahman W1, Rahimgir M2, Khan A A3, Khisa S4, Akter R5, Shahreen I6
DOI: https://doi.org/10.3329/jafmc.v15i1.48652

Abstract every year1. The hepatitis C virus is an RNA virus that belongs
to the family flaviviridae2. HCV replicates in the cytoplasm of
Introduction: The most common contemporary strategy to diagnose
hepatocytes, but is not directly cytopathic. Persistent infection
chronic hepatitis C virus (HCV) infection consists of initial screening
appears to rely on rapid production of virus and continuous
with an HCV enzyme immunoassay (EIA) antibody test followed by
cell-to-cell spread, along with a lack of vigorous T-cell immune
supplemental testing of positive screening tests with a quantitative
response to HCV antigens. The HCV turnover rate can be quite
HCV RNA assay to confirm the positive EIA and to determine
high with replication ranging between 1010 to 1012 virions per
whether they have active or resolved hepatitis C infection.
day, and a predicted viral half-life of 2 to 3 hours3. The rapid
Objectives: To detect and quantify HCV-RNA by real-time viral replication and lack of error proofreading by the viral RNA
polymerase chain reaction (real-time PCR) among anti-HCV polymerase are reasons why the HCV RNA genome mutates
positive patients and to identify the socio demographic factors frequently4. The transmission of HCV is primarily through
among these patients. exposure of infected blood. Risks for transmission include
blood transfusion, intravenous drug use, high risk sexual
Materials and Methods: This was a descriptive type of cross- activity, solid organ transplantation from an infected donor,
sectional study which was conducted in Combined Military occupational exposure, hemodialysis, household exposure
Hospital and Armed Forces Institute of Pathology, Dhaka and birth to an infected mother5. The aim of the present study
cantonment. A total of 108 anti-HCV positive patients by enzyme- was to find out HCV-RNA among anti- HCV positive (ELISA)
linked immunosorbent assay (ELISA), who were clinically patients by real time reverse transcriptase polymerase chain
suspected and advised for anti-HCV test, were selected randomly
reaction (RT-PCR) method.
for the study and subjected to do HCV-RNA analysis during the
period of October 2016 to September 2017. Materials and Methods
Results: Out of 108 anti-HCV positive patients by ELISA, HCV- This is a descriptive type of cross-sectional study which was
RNA was detected in 72 (66.7%) cases with mean value of conducted in Combined Military Hospital (CMH) and Armed Forces
HCV RNA quantification was 2013323.95±2695207.41 (IU/ ml). Institute of Pathology (AFIP), Dhaka cantonment through the
Majority of anti-HCV positive patients (29.6%) belonged to 51-
period from October 2016 to September 2017. Study population
60 years age group with male predominance (58.33%). It was
observed that 43.52% patients came from middle income group was anti-HCV antibody positive cases by ELISA of both sex and
family, 31.48% came from poor and 25.0% came from high all age groups. A total of 108 anti-HCV positive cases, who were
income group family. Risk factor for HCV infected population clinically suspected and advised for anti-HCV test, were selected
was found maximum in dialysis patients (47.37%), followed by randomly for the study. Relevant history was taken from the
blood transfusion (13.89%), Injecting drug User (IDUs) (12.04%), patients. Patients who were under treatment of HCV infection
surgery & intervention (9.26%) and sexual transmission (1.85%). and other liver diseases, who were suffering from hepatitis B
Mean alanine aminotransferase (ALT) was found 67.30±44.99 virus (HBV), human immunodeficiency virus (HIV) infection along
U/L among HCV-RNA detected patients (p< 0.05). with HCV infection and pregnant women, were excluded. Face
Conclusion: The quantification of HCV RNA by RT-PCR will be to face interview was done with the patients and a structural
helpful to rationalize the treatment, enhance antiviral responses questionnaire was used to collect data. Ethical clearance was
and mitigate mortalities of HCV infected patients. taken from Directorate General of Medical Services. With the
written consent of the patients HCV-RNA was extracted first and
Key-words: ELISA, HCV-RNA, Real time PCR then HCV-RNA was detected and quantified by real time reverse
transcriptase polymerase chain reaction (PCR) among anti-HCV
Introduction
positive patients.
Hepatitis C virus (HCV) infection is a serious global health
problem that affects 180 million people worldwide. It is Nucleic acid extraction: RNA was extracted from 200µl of plasma
estimated that three to four million people are infected with HCV with nucleic acid extraction kit and stored at -20℃ till used for PCR.

1. Maj Wasila Rahman, MBBS, MCPS, DCP, FCPS, Assistant Professor of Microbiology, Armed Forces Medical College (AFMC),
Dhaka (E-mail: wasila.rahman@yahoo.com) 2. Brig Gen Md Rahimgir, MBBS, MCPS, FCPS, MMEd, Ex-Professor and Head,
Department of Microbiology, AFMC, Dhaka 3. Brig Gen Arif Ahmed Khan, MBBS, MCPS, FCPS, Deputy Commandant, Armed Forces
Institute of Pathology (AFIP), Dhaka 4. Maj Suman Khisa, MBBS, MCPS, DCP, FCPS, Classified Specialist in Pathology, Border Guard
Hospital, Thakurgaon 5. Dr. Rahima Akter, MBBS, Officer on Special Duty, Director General of Health Services, Mohakhali, Dhaka,
6. Maj Imana Shahreen, MBBS, MCPS, Trainee Officer, AFIP, Dhaka.

84 JAFMC Bangladesh. Vol 15, No 1 (June) 2019

Detection and Quantification of HCV-RNA by RT-PCR among Anti-HCV Positive Patients

Real time reverse transcriptase polymerase chain reaction (RT-

PCR) for detection and quantification of HCV-RNA: The artus HCV
RG RT-PCR Kit constitutes a ready-to-use system for the detection
of HCV RNA using polymerase chain reaction (PCR) on Rotor-
Gene Q Instruments. The Hepatitis C Virus RG Master A and B
contain reagents and enzymes for the reverse transcription and
specific amplification of a 240bp region of the HCV genome, and
for the direct detection of the specific amplicon in fluorescence
channel Cycling Green of the Rotor-Gene Q. In addition, the artus
HCV RG RT-PCR Kit contains a second heterologous amplification
system to identify possible PCR inhibition. This is detected as an
Figure-1: Risk factors of the HCV transmission among study
internal control (IC) in fluorescence channel Cycling Orange of the
Rotor-Gene Q. The detection limit of the analytical HCV RT-PCR population (n=108)
is not reduced. External positive controls are also supplied, which
allowed the determination of the amount of viral RNA. Discussion
Enzyme linked immunosorbent assay test allowing the detection
Results
of anti-HCV antibodies has false positive results and require the
Age distribution of the anti-HCV positive patients was observed development of more sensitive and specific assays to confirm the
that out of 108 patients, majority 32(29.6%) patients belonged results. PCR, allowing the hepatitis C virus diagnosis by showing
to age 51-60 years and lowest number 6(5.6%) was at the age directly HCV RNA sequences, offers a complementary approach
of ≤20 years. Sex distribution was observed that 63(58.33%) to immunoserological tests6. Quantification of hepatitis C virus
patients were male and 45(41.67%) were female. Male to female (HCV) RNA is useful in clinical practice. Indeed, monitoring of
ratio was 1.4:1. In economic status of the HCV infected patients the fall in HCV RNA levels is presently used to decide whether
observed that majority 47(43.52%) patients were from middle to continue or stop therapy for patients. New directions in HCV
income group family, 34(31.48%) came from poor and 27(25.0%) therapy suggest that future treatments will be tailored to the
from high income group family (Table-I). Figure-1 shows that risk individual patient and that HCV RNA load monitoring during
factor for HCV infected population was found maximum in dialysis therapy will be a major treatment-tailoring tool. Thus, HCV RNA
patients (45.37%), followed by blood transfusion (13.89%), quantification assays need to be sensitive enough to detect
intravenous drug users (IDUs-12.04%), surgery and intervention HCV RNA reductions during therapy and also accurate in both
(9.26%) and sexual transmission (1.85%). the higher range (baseline viral load in untreated patients) and
the lower range (patients on therapy) of HCV RNA levels. This
In the present study, among 108 anti-HCV antibody positive can help to clarify the mechanisms underlying antiviral treatment
patients, 72(66.7%) were HCV-RNA positive by RT- efficacy and failure and derive recommendations on the basis of
PCR and the mean value of HCV-RNA quantification was
viral load monitoring for routine patient care. Thus, accurate HCV
2013323.95±2695207.41 (IU/ml), minimum was 75 (IU/ml) and
RNA quantification is absolutely crucial for hepatitis C therapy7.
maximum was 9962885.00 (IU/ml). In the present study, mean
ALT was 67.30±44.99 U/L among HCV-RNA positive patients and In the present study, 108 patients were found anti-HCV positive
36.72±34.76 U/L whose HCV-RNA was not detected or absent. by ELISA who were clinically suspected and advised for anti-HCV
That was statistically significant (p<0.05). Here, Out of 72 HCV- test by clinicians. Similar observation was found by Al-Mahtab
RNA positive cases, 31(43.05%) received dialysis. et al. in their study8. It was observed that out of 108 patients,
majority 32(29.6%) patients belonged to age 51-60 years and
Table-I: Socio-demographic characteristics of the study population lowest 6(5.6%) numbers belonged to age ≤ 20 years. Sixty three
(n=108) (58.33%) patients were male and 45(41.67%) were female. Male
to female ratio was 1.4 : 1. In a study of Al-Mahtab et al8 found that
Characteristics Frequency Percentage anti-HCV was positive in the age group between 17-50 years and
≤ 20 years 06 05.6 was male predominated 66% (6/9) over female 33%.
21-30 years 14 13.0
In this study, risk factor for HCV infected population was found
31-40 years 12 11.1
Age in years maximum in dialysis patients (45.37%), followed by blood
41-50 years 27 25.0
transfusion (13.89%), IDUs (12.04%), surgery and intervention
51-60 years 32 29.6 (9.26%) and sexual transmission (1.85%). Blood and its
> 60 years 17 15.7 components (plasma, serum, albumin etc) are the main source
Male 63 58.3 of HCV infection. In recent years, an increasing role has been
Sex Female 45 41.7 assigned to hospital transmitted infections (nosocomial)9. Rahman
et al in his study found 27.3% of the hemodialysis patients were
positive for anti HCV10. In present study, 72(66.7%) were HCV-
Poor income (<10,000TK) 34 31.48
Economic RNA positive tested by real time PCR among 108 ELISA positive
Middle income (10,000-40,000TK) 47 43.52 cases. Gao et al9 reported that the result-agreement rate of ELISA
status
High income (>40,000TK) 27 25.00 and real time PCR methods were 78.38%. It was observed in the
JAFMC Bangladesh. Vol 15, No 1 (June) 2019 85
 Detection and Quantification of HCV-RNA by RT-PCR among Anti-HCV Positive Patients

present study that the mean ALT was 67.30±44.99 U/L among 2. Lauer GM, Walker BD. Hepatitis C virus infection. N Engl J Med. 2001;
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TJ, et al. Hepatitis C viral dynamics in vivo and the antiviral efficacy of
Out of 72 HCV-RNA positive cases, 31(43.05%) received
interferon-alpha therapy. Science 1998; 282(5386): 103–7.
dialysis. A study carried out by Rahman study it was observed
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related deal10.
7. Karine Gourlain, Alexandre Soulier, Bertrand Pellegrin,Magali Bouvier-
Alias et al. Dynamic Range of Hepatitis C Virus RNA Quantification with
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The 2002 NIH consensus conference11 gave recommendations Microbioly 2005; 43(4):1669–73.
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concern the decision to treat, optimal treatment schedules, and
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important for clinical and therapeutic management of chronic infectors. Wei Sheng Yan Jiu 2007; 36(1):69-71.
HCV infection without additional costs.
10. Rahman AKMM. Hepatitis ‘B’ and Hepatitis ‘C’ Virus Infection in
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