The correlation between RT-PCR and ELISA assay on hepatitis C positive serum samples

Hepatitis C Virus is an infectious entity mainly affects liver. The HCV belongs to family Flaviviridae of viruses. The virion has single stranded positive polarity RNA genome. It was identified in 1989. It is transmitted through transfusion of infected blood, homosexuality, vertical transmission, inadequately sterilized surgical or dental instruments. Different techniques for diagnosis of HCV are available like ICT, ELISA, MEIA and RT-PCR etc. The objective of this study was to find out the most sensitive and reliable method for diagnosis of HCV infection. The study was conducted at a Public Sector Hospital, Hayat Abad Medical Complex Hayat Abad, Peshawar. A total of 300 samples tested for anti HCV antibodies on ELISA, out of which 14 (4.66%) were found positive to anti-HCV antibodies. These positive cases were considered for HCV genome detection using RT-PCR. The results showed that 05 (1.66%) samples are positive for HCV RNA while 09 were found negative. It is determined that RT-PCR technique can be used for the detection of Hepatitis C Virus because it is quick and reliable.


Introduction
Hepatitis C is an infectious viral disease of liver.The infection is caused by Hepatitis C virus.HCV belongs to Flaviviridae family of viruses, compressed of about 9.6 kb single stranded positive sense RNA genome [1].Based on genotypes, six major HCV strains and more than fifty subtypes have been identified so far [2].The disease is often asymptomatic initially, however, infection with HCV can lead to chronic liver disease in which scarring of liver can occur and if it continues to progress then cirrhosis and in some cases hepatocellular carcinoma is the end result that appears after years [3].The HCV infection is not confined to a limited area, the prevalence is all over the world, rising health, economic and social issues [3,4].The prevalence is directly proportional to the developed and developing regions as the developed countries has proper hygiene and better protection measures as compared to developing regions.The overall prevalence of hepatitis C infection contamination in the world is evaluated by the World Health Organization to be around 3%.This corresponds to about 170 million persons are infected throughout the globe.According to WHO recent reports, three to four million new cases diagnosed and more than 0.

Materials and methods
The study was conducted at the department of pathology, Hayat Abad Medical Complex Hayat Abad, Peshawar during March to June 2015.The purpose of the entire study was to identify the correlation between the serological and nucleic acid base diagnosis of HCV.The informed consent was obtained from the patients before sample collection.

Laboratory methods ELISA
A total of 300 blood samples were screened for the presence of Anti-HCV antibodies through third generation ELISA [Biokit, Barcelona SA, Spain].Serum was separated from 5 ml of blood in the disposable tubes.Further process were followed according to the manufacturers instructions ELISA (Biokit, Barcelona SA, Spain).

Real Time PCR and Extraction of RNA
The anti HCV positive cases were further processed for the detection of RNA using Sacace (Sacace, Biotechnology, Italy) extraction and RT-PCR kit.Cepheid smart cycler (Nasdaq: CPHD, California, US) was used for the amplification process.

Results and discussion
Three hundred blood samples were screened for the presence of anti antibodies.These samples were collected from the patients who have visited the hospital purposely.Out of 300 blood samples 14 (4.66%) were found positive for Hepatitis C Virus antibodies on ELISA.The positivity of the cases was considered according to the defined OD value.The positive cases to anti HCV antibodies, were further analyzed for the presence of RNA.Among these 14, 09 cases were found negative for HCV RNA, while 05 (1.66%) cases were found active to the infection (Table 2).

Figure 1 .
Figure 1.Correlation between ELISA and RT-PCR resultsConclusion and recommendationsThe study has provided us knowledge of the importance of diagnosis of the disease prior to therapy.The following points should be taken into consideration.Initial diagnosis may be carried serologically but the positive cases must be confirmed with nucleic acid based technique.The persons with Hepatitis symptoms should be tested for infection.The immunocompromised patients having liver diseases should be tested for infection.Authors' contributionsConceived and designed the experiments: N Bahadar & S Ahmad, Performed the experiments: N Bahdar, M Israr & F Khan, Analyzed the data: F Khan & M Israr, Wrote the paper: N Bahadar Acknowledgment The authors are thankful to Mr. Noor Rehman for his unconditionl guidance.We are also thankful to Mr. Naveed and Mr. Izhar Ullah for providing the proper guidance in the laboratory during the work.