GREENER JOURNAL OF MEDICAL SCIENCES
ISSN: 2276-7797 ICV: 5.98
Submitted: 13/02/2017 Accepted: 16/02/2017 Published: 28/03/2017
Research Article (DOI: http://doi.org/10.15580/GJMS.2017.2.021317025)
Molecular Epidemiology of HCV Genotype in Relation to Viral Load of Infected Individuals in Northwestern Nigeria
*1Umar Faruk Abubakar, 2Muhammad Yahaya, 3Sumayya Hamza Maishanu, 2Ismail Ibrahim, 3Aisha Rabiu Ishaq, 4Alo Moses Nnaemeka, 1Abdullahi Sani Ahmad, 5Muhammad Yahaya
1*Public Health and Diagnostic Institute, Northwest University Kano, Nigeria
2Faculty of Medical Laboratory Science, Usmanu Danfodiyo University Sokoto, Nigeria
3DNA Labs, Molecular Research and Diagnostic Center Kaduna, Nigeria
4Department of Biological Science, Federal University, Ndufu-Alike, Ebonyi State, Nigeria
5Laboratory Department, Rasheed Shekoni Specialist Hospital, Dutse, Jigawa, Nigeria
*Corresponding Author’s Email: Farouqabu @gmail .com
ABSTRACTS
The global burden of Hepatitis C Virus (HCV) is increasing and its believed to be one of the leading causes of hepatocellular carcinoma and a major cause of chronic liver disease. The present study aimed at evaluating the prevalence of HCV genotype in association with viral load of infected individuals. A total of one hundred and seventy three (173) HCV seropositive patients were included in the study. RNA extraction was performed using Accuprep RNA extraction kit according to the manufacturer’s instructions.After running the HCV cDNA on 1.5% agarose gel, genotyping was completed using Beckman Coulter sequencing machine based on chain termination method.Accupower HCV-1111 (IVD) was used for the preparation of master mix and light cycler real time PCR machine version 5.2 was used for viral load. The result of the current study revealed that 93 (54%) patients were infected with the viral genotype 1b, followed by 1a with 64 (39%), 1c, 2a and 2b with 2 (1%), 9 (5%) and 5 (3%) respectively. Conclusively, genotype 1b has the highest prevalence, while 1c with lowest number of infected individuals.Moreover, in all the ranges of viral loads in the study, 1b was found to have highest prevalence.
Key Words: HCV, Viral load, Genotype.
1. INTRODUCTION
Hepatitis C Virus (HCV) is one of the number one causes of hepatocellularcarcinoma and a major cause of chronic liver disease (Seeffet al., 2001; Houghton 1996), it has a high mortality rate which account for approximately 350000 deaths of the infected people every year.It is estimated that about 3% of the world’s population have HCV. There are about 4 million carriers in Europe alone (Jinget al.,2015). HCV is estimated to be ten times more infectious than Human Immunodeficiency virus (HIV) but less infectious than Hepatitis B virus (Seeffet al., 2001). Hepatitis C virus was discovered in 1989 during the era of research aimed at finding the agent responsible for 80% of transfusion associated hepatitis cases other than hepatitis A and B which as of then was called non-A, non-B hepatitis and the presumed etiologic agent of non-A non-B hepatitis virus (Hsuet al.,1994; Alter1999). HCV is positive sense single strand RNA virus of about 50nm in diameter belonging to the genus Hepacivirus and a member of the Falviviridae family (Mackay 2004; Hsuet al.,1994; Alter1999). It is spherical, lipidenveloped with genome of about 9600 nucleotidesand six major genotypes (1-6) have been identified to date (Tsukiyama-Koharaet al.,1992). The genotypes differ by 31 to 34% in their nucleotide sequences based on their full-length genome sequence comparisons (Chooet al.,1985;Simmondset al.,2005).Transmission is mostly by blood and blood products, individuals that use intravenous drugs are equally at risk of infection. In addition, vertical transmission and sex with an infected partner are also risk factors (Dusheikoet al.,2005; Esfahaniet al.,2010). HCV infection is generally asymptomatic during the acute phase and about 85% of them become chronically infected.HCVtherapy is genotype/subtype specific which makes the genotype identification crucial for clinicians when choosing chemotherapy, because these genotypes have been reported to exhibit different responses to prescribed anti-viral therapies and require varying duration and doses of therapy (Jimenez-Mendezet al.,2010). Its genotype is worldwide distributed but some strains are peculiar to some particular geographical regions, 1a, 1b and 3a genotypes are prevalent strains all over the world (Pawlotsky, 2003). Genotypes 1, 2 and 4 are confined to certain regions of Africa and the Middle East, whereas genotypes 3 and 6, divergent endemic strains are detected in numerous localities of Southeast Asia (Purcell, 1994).
2. MATERIALS AND METHODS
Study Area: the study was conducted in DNA Labs Kaduna, Northwest geopolitical zone of Nigeria. It is a facility that receives samples for HCV viral load and genotyping from all the seven states of northwest geopolitical zone (Kaduna, Kebbi, Kano, Katsina Zamfara, Jigawa and Sokoto) which makes it suitable for this research.
Subjects: the study comprised of 173 HCV antibody positive patients that requested for HCV viral load and genotype from any of the seven states in northwest geopolitical zone.
Inclusion and Exclusion Criteria: the inclusion criteria comprises of hepatitis C antibody positive patients of all sex and age. The study excludes patients outside northwest geopolitical zone or hepatitis C seronegative patients.
Specimen Collection:blood samples were collected by standard venopuncture without undue pressure either on arm or the syringe and dispensed in a plain container. It was allowed to clot for 30min and spun at 10,000 rpm for 5minutes in order to obtain serum. The separated sera were stored at -200C until used for analysis.
Viral RNA Extraction: the extraction was conducted using Accuprep® viral RNA extraction kit. Binding buffer was used to lyse the cells in the serum followed by alcohol precipitation. The precipitated proteinwas filtered using a binding column, washed and eluted to obtain the RNA.
Master Mix and Viral Load Preparation: Accupower HCV-1111 (IVD) was used for the preparation of master mix. HCV lyophilized premix was reconstituted with ultra-pure water and eluted RNA was added. The mixture was taken immediate into a real time PCR machine (Roche) light cycler for the reverse transcription (cDNA) and viral load was ascertain upon completion of the run.
Genotyping: reconstituted RT-PCR premix was mixed with a specific volume of the eluted RNA and cDNA synthesis was performed at 50°C for 1 hour. Nested PCR was conducted using UTR1 and UTR2 primers for the 1st round, while UTR3 and UTR4 primers were used for the 2nd round, using the conditions (94°C for 1 minutes, 94°C 25 for seconds, 44°C for 40 seconds, 72°C for 1 minute and 72°C for 10 minutes) and (95°C for 1 minute 95°C for 20 seconds, 53°C for 30 seconds, 72°C for 25 seconds and 72°C for 10 minutes) respectively. The PCR product was ran on a 1.5% agarose gel at 120 volt, band size was compared with the molecular ladder by placing the gel on a UV light source and the right band was carefully cut for the gel extraction. Upon completion of the gel extraction, sequencing reaction was performed using the 2nd round reverse primer (UTR4) and the cycling was repeated 30 times. The sequencing reaction product was cleaned up and taken to the Beckman Coulter sequencing machine, obtaining the electrophorogram at the end of the run. The sequence was generated and BLASTED accordingly on GENEBANK to ascertain the HCV genotype.
3. RESULTS
4. DISCUSSION
Hepatitis C viral disease plays a negative impact on the lives of many people globally, the distribution of its genotypes vary with respect to geographical location. Genotypes 1-3 being the worldwide distributed genotypes (Dusheikoet al.,2005; Esfahaniet al.,2010).
The current study revealed that genotypes 1 and 2 were found to be the major causes of the disease in Northwestern part of Nigeria; with the subtype 1b having the highest number of patients (54%) and only (1%) have been found to be subtype 1c while 1a, 2a and 2b were found to have (37%), (05%) and (03%)respectively.A similar research by Jinget al., (2015)shows a predominant percentage of subtype 1b among the test subjects, which is in support of the present study. This finding contradict with the study conducted by Indian author Anita Charkravatiet al., (2011)and Pakistan author Sulimanet al.,(2014)where genotype 3a was found to be the predominant subtype.It is however in contrast to the findings of Xieet al., (2016) and Ederthet al., (2016) that found 1a to be the subtype with highest percentage in USA and Sweden respectively.Findings by Joseph et al., (2015)also found genotype 2 as the leading cause of HCV in Ghana accounting for about 80% of HCV cases.
The total of 48 patients with viral load < 200000IU/ml was recorded, with the genotype 1b taking the highest number of patients, 27 (56.3%) while 1a has 19 (39.6%) number of patients and 2 (4.2%) patients represent 2b genotype. None of patients was found with the genotype 1c and 2a.
In the category of viral load of 200001 – 400000IU/ml, the study revealed a 0% patient with HCV genotype 2b among the 25 patients within the range. Eleven (44%), 9 (36%), 2 (8%) and 3 (12%) patients were found to be infected with the viral genotype 1b, 1a, 1c and 2a respectively.
Out of the 71 HCV infected individuals within the range of 400001 – 600000IU/ml viral load, 39(54.9%) patients have 1b genotype, followed by 1a with 26 (36.6%) while 4 (5.6%) patients were found to have 2a and genotype 2b has 2 (2.8%) number of patients but there was no patient with 1c.
Among the 29 patients with viral load > 600000IU/ml, genotype 1b has the highest prevalence of 16 (55.2%). There was no patient with HCV genotype 1c while 1a, 2a and 2b were found to have 10 (34.5%), 2 (6.7%) and 1 (3.4%) patients respectively.
5. CONCLUSION
In conclusion, the findings of the present study revealed that 93 (54%) patients were infected with genotype 1b, followed by 1a with 64 (39%), 1c, 2a and 2b with 2 (1%), 9 (5%) and 5 (3%) respectively. However, genotype 1b was found to have the highest prevalence, while 1c with lowest number of infected individuals. Moreover, in all the ranges of viral loads in the study, 1b genotype was found to have highest prevalence.
6. RECOMMENDATION
From the current study, it is recommended that the genotype and viral load of the virus should be known before commencement of therapy for any HCV positive patient and also to ensure effective management of the patients, viral load should be monitored accordingly.
7. ACKNOWLEDGEMENT
The author acknowledge the assistance of DNA labs research and diagnostic laboratory Kaduna, particularly management and technical staff
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Cite this Article: Umar FA, Muhammad Y, Sumayya HM, Ismail I, Aisha RI, Alo MN, Abdullahi SA, Muhammad Y (2017). Molecular Epidemiology of HCV Genotype in Relation to Viral Load of Infected Individuals in Northwestern Nigeria. Greener Journal of Medical Sciences, 7(2): 018-021, http://doi.org/10.15580/GJMS.2017.2.021317025