1. Oladeinde Bankole Henry Reg no: 2012617004F
Molecular Epidemiology of Hepatitis C Virus among HIV infected patients in Benin City, Nigeria By
Oladeinde Bankole Henry
Reg no: F
Department of Medical Laboratory Science
Faculty of Health Sciences and Technology
Nnamdi Azikiwe University, Awka , Nigeria
Supervisor: Professor . I. M Ekejindu
March 2014

2. INTRODUCTION
The Human immunodeficiency Virus (HIV) infection is associated with increased mortality and morbidity worldwide (Monga et al., 2001)
Sub- Saharan Africa remains by far the most affected region with about 24.5 million people living with HIV, representing a little below two-thirds of all people living with HIV in the world(Oladeinde et al., 2011 )
HIV infection is a major health concern in Nigeria, where it is estimated that about 2.9 million people are living with the virus (Monjok et al., 2009 ).

3. INTRODUCTION CONTINUED
Hepatitis C virus (HCV) infection is a major global public health problem in both developed and developing countries (Jadim et al., 2013).
HCV is reported to be the leading cause of chronic liver disease worldwide (Qui et al., 2009).
WHO estimates that about 3% of the worlds population are infected with HCV (WHO, 1999).
With a prevalence of 5.3% and an estimated 32 million people infected with HCV, Sub-saharan Africa has the highest burden of the disease in the world (WHO, 1999 ; Karoney and Silka, 2013).
Risk factors for HCV infection (Oladeinde et al., 2012; Shaikh et al., 2009; Kandeel et al., 2012; Opalskalki and Kovac, 2011; Robert et al., 2012).

4. INTRODUCTION CONTINUED
HCV is spread 10 times more efficiently than HIV through percutaneous blood exposures ( Gupta, 2013)
HCV structure and genotypes (Qui et al., 2009)
As a result of shared routes of transmission, co- infection with hepatitis C virus (HCV) is common among HIV – infected patients (Sulkowski, 2013).
HIV-HCV co-infection is associated with significant morbidity and mortality (Mboto et al., 2013).
Of HIV-infected individuals in developed countries of the world, 20%–30% are co-infected with hepatitis C virus (Shuhart et al., 2006).

5. INTRODUCTION CONTINUED
While approximately 20% of HCV mono-infected patients clear the virus, clearance of HCV occur in less than 10% of HIV-HCV co-infected patients, especially in patients with lowered CD4+ count (Gupta, 2013).
Several studies have confirmed that HIV co-infection accelerates the clinical course of chronic HCV infection leading to an increased risk of cirrhosis, and hepatocellular carcinoma (Ying et al., 2009)
However conflicting reports exists on the role of HCV on the progression of HIV infection to AIDS (Babik and Holodniy, 2003).

6. INTRODUCTION CONTINUED
Daar et al., 2001 and Greub et al., 2000 reported that co- infection with HCV confers an increased risk for progression to AIDS in HIV-infected individuals. Other studies, however failed to demonstrate that increased risk (Wright et al., 1994; Rockstroli et al ., 2005)

7. JUSTIFICATION OF STUDY
HCV is reported to be common among HIV infected patients (Sulkowski 2013). Nigeria is reported to be the third most endemic HIV nation in the world (Monjok et al., 2009).
Few studies presently exist on prevalence of HCV infection among HIV infected patients in Nigeria.
HCV related liver disease has emerged as a major cause of mortality and morbidity among HIV infected patients since the advent of Highly Active Antiretroviral Therapy (HAART) (Salmon – ceron et al., 2005).

8. JUSTIFICATION OF STUDY CONTINUED
Conflicting reports exists on role of HCV infection on progression of HIV disease progression (Babik and Holodniy, ).
No study presently exist on the effect of HCV infection on HIV disease progression in Nigeria.
HCV genotype influences both response to therapy and disease severity as well as the viral-host interactions (Moreau et al., 2008).
No study have reported the circulating HCV genotypes and subtypes among HIV infected patients in Nigeria.
Due to the geographic clustering of distinct HCV genotypes, genotyping may be a useful tool for tracing the source of an HCV outbreak in a given population (Zein et al., 2000).

9. AIM OF STUDY
To determine the prevalence and distribution of
HCV genotypes/subtypes among HIV infected
patients in Benin City, Nigeria.

10. SPECIFIC OBJECTIVES
To determine the sero-prevalence of HCV infection among HIV infected patients in Benin City, Nigeria
To determine the effect of age, gender, history of blood transfusion and other demographic factors on sero- prevalence of HCV among HIV infected patients in Benin City, Nigeria
To determine the effect of HCV infection on the immunity of HIV infected patients using CD4+ count as immunological marker.
To determine the association between HCV genotypes and CD4+ count of HIV infected patients.

11. MATERIALS AND METHODS
Study Area:
This study will be conducted at the University of Benin Teaching Hospital, a tertiary health institution in Edo State, Nigeria
Institutional approval for study
Study approval for this work will be sought and obtained from the Ethical Committee of the University of Benin Teaching Hospital, Benin City, Edo State, Nigeria.

12. SAMPLE SIZE DETERMINATION
The minimum sample size for this study will be obtained using the formula
N = Z2 * P(1-P)/D (Naing et al., 2006)
Where,
N = Minimum sample size
D = Desired level of Significance (set at 0.05)
Z = Confidence Interval (1.96)
P = Prevalence rate (6.5%) in a target population from previous study (Taye and latew, 2013)
Computation with the values above will give a sample size of 94.

13. Study Participants: Two sets of persons will be used for this study.
STUDY POPULATION
Study Participants: Two sets of persons will be used for this study.
Group A – Consisting of 500 HIV sero-positive patients (Test group).
Group B – Consisting of 250 apparently healthy HIV sero- negative subjects (Control group).
Ethical Issues
Informed consent will be obtained from all participating subjects of study before commencement of work. .

14. STUDY POPULATION CONTINUED
In case of minors, informed consent will be obtained from parents/guardians before commencement of work.
INCLUSION CRITERIA FOR TEST GROUP: HIV sero- positive patients registered at the University of Benin Teaching Hospital, Benin City.
INCLUSION CRITERIA FOR CONTROL GROUP: HIV sero-negative asymptomatic persons
EXCLUSION CRITERIA FOR CONTROL AND TEST GROUPS: Persons less or equal to 18 months of age.

15. STUDY DESIGN
This is a cross-sectional study.

16. SPECIMEN COLLECTION
Ten milliliters of venous blood shall be obtained from each HIV consenting patients of study. Five millilitres of blood each will be dispensed into two ethylene diamine tetra-acetic acid (EDTA) containers and mixed.
Five millilitres of blood will be collected from apparently healthy persons and placed in an EDTA container and mixed.
All tubes will be appropriately labeled.

17. PROCESSING OF SPECIMEN
All samples collected from patients and control groups will be screened for presence of HIV antibodies using current national algorithm for HIV sero-diagnosis.
All HIV positive patients will be tested for antibodies for Hepatitis C Virus using immunochromatographic methods (Clinotech Diagnostics, Richmond, Canada and Orient Gene Biotech China).
This will also be done for all HIV sero- negative control subjects.

18. Processing of specimen continued
1.5 mililitres of plasma from all HCV / HIV sero positive patients will be dispensed into greiner bio-one cryovials and stored at -80 oC.
Same volume of plasma will be stored from all control subjects that tested positive to HCV antibodies.
These stored plasma will subsequently be used for genotyping and subtyping of HCV.
CD4+ T lymphocyte estimation will be done on all HIV infected patients using flow cytometry method (Partec, GmbH, Germany).

19. Processing of specimen continued
The second 5.0 mililitre EDTA blood will be used for this estimation.
HCV-RNA extraction
HCV- RNA will be extracted from 140µl of stored anti- HCV positive plasma using the Qiagen Viral RNA mini kit (Qiagen Hilden, Germany).
cDNA synthesis
cDNA synthesis will be carried out from HCV-RNA extract using avian myeloblastosis virus reverse transcriptase (AMVRT) . This will be carried out using

20. PROCESSING OF SPECIMEN CONTINUED
µl of RNA template
2. 10 µl of free RNase Water
3. 2 µl Oligo(dT) Primer
4. Incubate mixture at 65OC for 2mins in a thermocycler
5. Chill on ice for 1minute.
While still on ice , add
a. 2.0 µl AMVRT reaction buffer
b µl Dithiothreitol (DTT)
c µl dNTP

21. PROCESSING OF SPECIMEN CONTINUED
d. 0.5 µl RNase inhibitor e. 1.0 µl Avian Myeloblastosis Virus Reverse transcriptase 6. Mix reaction tube gently and incubate at 37OC for 60 minutes 7. Terminate reaction by heating at 85OC for 5 minutes 8. Chill on ice for 1 minute

22. PROCESSING OF SPECIMEN CONTINUED
Nested PCR amplification of the 5` UTR
This will be carried out with
pmol of each reverse and forward primers
2. 0.5ml Phusion HF Buffer
Mm dNTPs
U of Tag DNA polymerase
5. 50mM Potassium chloride
The reaction will be performed in thermocycler and programmed as follows
1. 94OC for 25 secs
2. 50OC for 40 secs
3. 72OC for 60 secs and 72OC for 5mins. This will be repeated for 25 cycles

23. PROCESSING OF SPECIMEN CONTINUED
Second round PCR amplification
1.0µl of the first round amplicon will be re-amplified using internal primers for another 25 cycles under the same conditions.
Table 1: SHOWING PRIMERS FOR 1ST AND 2ND ROUND AMPLIFICATION PROCESS
Table 1
PCR round
Primer sequence
Polarity
1st round
5`-CTGTGAGGAACTACTGTCTT -3`
5`- ATACTCGAGGTGCACGGTCTACGAGACCT-3`
outer sense
outer anti-sense
2nd round
5`TTCACGCAGAAAGCGTCTAG-3`
5`-CACTCTCGAGCACCCTATCAGGCAGT-3`
Outer anti-sense

24. PROCESSING OF SPECIMEN CONTINUED
The amplicons will be analyzed on 2% gel followed by staining with ethidium bromide, and visualized under a Ultraviolet transluminator.
A molecular weight marker of 250 bp will be used.
Purified PCR products from above will be sent to Inquaba Diagnostics South Africa for sequencing
The sequences obtained will be compared with existing HCV sequences in the GenBank to know the HCV genotype and subtypes.

25. RESULTS
The results obtained will be organized and subjected to appropriate statistical analysis

26. STATISTICAL ANALYSIS
The data obtained will be analyzed using Chi square (X2) test and odd ratio analysis using the statistical software INSTAT®. (Graphpad software Inc., La Jolla, CA, USA). Statistical significance was set at P < 0.05.

27. DISCUSSION AND CONCLUSION
Findings observed in this study shall be discussed by comparison with other available literature and appropriate conclusions will be drawn from it.

28. REFERENCES
Babik, JM and Holodniy M. (2003). Impact of Highly Active Antiretroviral Therapy and Immunologic Status on Hepatitis C Virus Quasispecies Diversity in Human Immunodeficiency Virus/Hepatitis C Virus- co-infected Patients. Journal of Virology 77(3): 1940–1950.
Center for Disease Prevention and Control (1998). Recommendations for Prevention and Control of Hepatitis C Virus (HCV) Infection and HCV-Related Chronic Disease. Morbidity and Mortality Weekly Report 47(19): 1-39.
Daar ES, Lynn H, Donfield S, Gomperts E, O’Brien SJ, Hilgartner MW, Hoots WK, Chernoff D, Arkin S,Wong WY, Winkler CA, and the Hemophilia Growth and Development Study (2001). Hepatitis C virus load is associated with human immunodeficiency virus type 1 disease progression in hemophiliacs. Journal of Infectious Disease 183:589–595.
Greub G, Ledergerber B, Battegay M, Grob P, Perrin L, Furrer H, Burgisser P, Erb P, Boggian K, Piffaretti JC, Hirschel B, Janin P, Francioli P, Flepp M, Telenti A (2000). Clinical progression, survival, and immune recovery during antiretroviral therapy in patients with HIV-1 and hepatitis C virus coinfection: the Swiss HIV Cohort Study. Lancet 356:1800–1805

29. REFERENCES CONTINUED
Gupta P (2013). Hepatitis C Virus and HIV type 1 co-infection. Infectious Disease Reports 5: 1-7
Ibrahim AA, Haseeb AK, Ali HB,Ali AA, Ahmed HA, Mohamad S, Mohamad AS (2009). Comparison of neighbor-joining and maximum- parsimony methods for molecular phylogeny of Oryx species using 12S RRNA nad 16S RRNA gene sequence. Journal of Animal Biology 1(2): 1-9.
Jardim ACG, Bittar C, Matos RPA, Yamasaki LHT, Silva R, Pinho JRR, Fachini RM, Carareto CMA, de Carvalho-Mello IMVG, Rahal P (2013). Analysis of HCV quasispecies dynamic under selective pressure of combined therapy. BMC Infectious Disease 13:(61).
Kandeel AM, Talaat M, Afifi SA, El-Sayed NM, Fadeel MAA, Hajjeh RA, Mahoney FJ (2012). Case control study to identify risk factors for acute hepatitis C virus infection in Egypt. BMC Infectious Disease 12:(294).

30. REFERENCES CONTINUED
Karoney MJ and Siika AM (2013). Hepatitis C virus infection in Africa: a review. The Pan African Medical Journal 14: (44).
Mboto CI, Fielder M, Davies-Russell A, Jewell AP (2010). Co- infection with human immunodeficiency virus (HIV) is associated with significant morbidity and mortality. Hepatitis C virus prevalence and serotypes associated with HIV in The Gambia. British Journal of Biomedical Science 67(3):
Monga HK, Rodriguez-Barradas MC, Breaux K, Khattak K, Troisi CL, Velez M, Yoffe B (2001). Hepatitis C virus infection-related morbidity and mortality among patients with human immunodeficiency virus infection. Clinical Infectious Disease 33:

31. REFERENCES CONTINUED
Monjok E, Smesny A, Essien EJ (2009). HIV/AIDS - Related Stigma and Discrimination in Nigeria: Review of Research Studies and future directions for Prevention Strategies. African Journal of Reproductive Health 13(3): 21–35.
Moreau I, Levis J, Crosbie O, Kenny-Walsh E, Fanning LJ (2008). Correlation between pre-treatment quasispecies complexity and treatment outcome in chronic HCV genotype 3a. Virology Journal 5:78.
Naing L, Winn T, Rush BN (2006). Practical issues in calculating sample size for prevalence studies. Archives of Orofacial Sciences (1):9-14.
Oladeine BH, Omoregie R, Olley N, Anunibe JA (2011). Prevalence of HIV and aneamia among pregnant women. North American Journal of Medical Science 3: 548 – 551.

32. REFERENCES CONTINUED
Oladeinde BH, Omoregie R. Olley M, Anunibe JA, Oladeinde OB (2012). Hepatitis B and C viral infections among pregnant women in a rural community of Nigeria. International Journal of Basic and Applied Virology 1(1):1-5.
Oladeinde BH, Omoregie R, Oladeinde OB (2013). Prevalence of HIV, HBV and HCV infections among pregnant women receiving ante-natal care at a traditional birth home in Benin City, Nigeria. Saudi Journal of Health Sciences 2(2):
Operskalski E and Kovacs A (2011). HIV/HCV Co-infection: Pathogenesis, Clinical Complications, Treatment, and New Therapeutic Technologies. Current HIV/AIDS Report 8(1): 12–22.
Qui P, Cai X, Ding W, Zhang Q, Norris E, Greene JR (2009). HCV genotyping using statistical classification approach. Journal of Biomedical Sciences 16: 62

33. REFERENCES CONTINUED
Robert A, Weinstein F, Rania A, Scott D (2012). Transmission of hepatitis C virus infection through tattooing and piercing: A Critical Review. Clinical Infectious Disease 54 (8):
Rockstrock JK, Mocroft A, Soriano V, Turai C, Losso MH, Horban A, Kirk O, Phillip A, Ledergerber B, Lundgren L, EuroSIDA study group (2005). Influence of hepatitis C virus infection on HIV1 disease progression and response to highly active antiretroviral therapy. Journal of Infectious Diseases 192(6):
Salmon-Ceron D, Rosenthal E, Lewden C, Bouteloup V, May T, Burty C (2009). Emerging role of hepatocellular carcinoma among liver-related causes of deaths in HIV-infected patients: The French national Mortality study. Journal of Hepatology 50:
Shaikh F, Qaiser H, Naqvi H, Jilani K, Allah R, Memon D. (2009). Prevalence and risk factors for hepatitis C virus during pregnancy. Gomal Journal of. Medical Science 7(2):

34. REFRENCES CONTINUED
Shuhart MC, Sullivan DG, Bekele K, Hamintgton RD, Kitahata MM, Mathisen TL, Thomasson LV, Emerson SS, Gretch DR (2006). HIV Infection and Antiretroviral Therapy: Effect on Hepatitis C Virus Quasispecies Variability. Journal of Infectious Diseases 193 (9):
Sulkowski MS (2013). Current Management of Hepatitis C Virus infection in patients with HIV o-infection. Journal of Infectious Diseases (1):
Taye S and Lekew M (2013). Impact of hepatitis C virus co-infection on HIV patients before and after highly active antiretroviral therapy: an immunological and clinical chemistry observation, Addis Ababa, Ethiopia. BMC Immunology 14: 23

35. REFERENCES CONTINUED
WHO (1999): Hepatitis C- global prevalence (update). Weekly epidemiology record 49:
Wright TL, Hollander H, Pu X, Held MJ, Lipson P, Quan S, Polito A, Thaler MM, Bacchetti P, Scharschimdt BF (1994). Hepatitis C in HIV- infected patients with and without AIDS: prevalence and relationship to patient survival. Hepatology 20:1152–1155.
Ying L, Robinson M, Fu-jie Z. Human immunodeficiency virus and hepatitis C virus co-infection: epidemiology, natural history and the situation in China. Chinese Medical Journal 2009; 122(1):93-97.
Zein NN (2000). Clinical significance of hepatitis C virus genotypes. Clinical Microbiology Review 13:

36. INFORMED CONSENT FORM ----------------------- -----------------------
TITLE OF STUDY: MOLECULAR EPIDEMIOLOGY OF HEPATITIS C VIRUS AMONG HIV INFECTED PATIENTS IN BENIN CITY, NIGERIA.
  PURPOSE OF RESEARCH: The purpose of this study is to determine the prevalence of Hepatitis C Viral infection, HCV genotypes and subtypes among HIV infected patients as well as association between HCV and HIV disease progression. 
PROCEDURES INVOLVED IN THE STUDY: Blood will be collected from you to determine the presence of Hepatitis C virus as well as HIV status for research purpose only.
  COMPENSATION: There will be no financial compensation for participating in this study.
  VOLUNTARY PARTICIPATION: Please note that your participation in this study is entirely voluntary and no form of force will be used on you nor any form of discrimination meted out on you. On the event that you decide to stop participating, you are very free to withdraw even if you had earlier agreed to participate.
  RISKS: Blood will be collected from you. There is a little measure of painful discomfort associated with the blood collection. No other adverse effect or risk is expected to be associated with participation in this study.
   BENEFITS: Findings from this study will improve epidemiological knowledge of co-infection of HCV and HIV as well as improve the way Physicians will manage patients infected with HCV.
  CONFIDENTIALITY: Information obtained will be treated with utmost confidentiality. Your names will not be used. The blood samples will not be given out to other investigators for another study.
Please note that appending your signature to this from indicates that it has been read and you agree to participate in the exercise. You can contact me at or on the Telephone number
Signature Date

37. QUESTIONNAIRE
This questionnaire seeks to establish factors associated with HCV infection among study population. Please your candid opinion on questions asked is highly solicited. Tick and write where appropriate. Patients confidentiality will be strictly maintained.
Age
Gender ■ Male ■ Female
Place of Residence ■ Urban ■ Rural
Occupation
Highest educational level attained ■ No formal education ■ Primary ■ Secondary ■ Tertiary
Marital status ■ Married ■ Single ■ Widow
Do you have a child/children ■ Yes ■ No
If yes, where were they delivered?.
■ In an hospital ■ In a traditional birth centre ■ At home ■ A mixture of the afore –mentioned. Please specify
Ever been transfused with blood or blood products?. ■ Yes ■ No
Ever had surgery? ■ Yes ■ No
Do you have tattoos / body marks or facial marks ■ Yes ■ No
Please indicate the particular type of mark
Do you inject self with medications? ■ Yes ■ No
Have you started receiving treatment with HAART?. ■ Yes ■ No
If yes, for how long now?