Characterization of Hepatitis C Virus Circulating among Injecting Drug Users (IDU) in Kilifi County, Kenya.

Background: Hepatitis C virus is a major global health problem estimated to infect over 170 million people globally with the most common route of infection being injecting drug use (IDU). Treatment for HCV infection has traditionally been shown to be genotype specific; however the available drugs are still expensive and out of reach in many developing countries. To improve on monitoring, there is need to continuously document the genotypic burden and epidemiology in different populations arises. Objectives: This study aimed to determine the circulating genotypes and link the data to the socio-demographics of injecting drug users in Kilifi County along the Kenyan coastline. Methods: Using a random sampling method, this was a cross-sectional prospective study conducted among 127 injecting drug users, whereby ethical clearance was sought from Kenyatta National Hospital/ University of Nairobi Ethical and Research review committee (KNH/UON-ERC), and Reference number P366/07/2017 on 25 th September 2017. Serology for HCV was done followed by nucleic acid amplification and eventual genotyping. Sociodemographic data was collected using questionnaire administered at the sites. Results: A total of 28 (23 males and 5 females) samples out of 127 samples were positive for HCV giving a prevalence of 22.1%. The modal age group was 25- 49 years. Of the positive samples, 11 were amplified by PCR, all from the male IDUs. Prevalent genotypes included genotype 1a (13%) and genotype 4a (87%). Both genotype 1a and 4a were only found in males. Conclusion: Although it is the first time to report HCV4a in Kilifi town and Mtwapa as compared to Watamu and Malindi which had both HCV1a and HCV4a. Tourists born in countries where HCV prevalence is high are supposed to be screened before accessing entry to Kilifi County. No clinical trial was conducted. The study reveals the burden of HCV infection among IDUs in Kilifi County. The Government should formulate policies for c

intervention on testing and treatment of HCV in Kilifi County targeting IDUs in order to minimize spread to other populations.

Background
Hepatitis C virus causes of liver cancer such as hepatocellular carcinoma (HCC) and lymphomas in humans (Ferri 2015and Rusyn 2014). Hepatitis C is a global health problem, over130 million people are chronically infected yearly (Amal 2015). Studies show that every year, approximately 308,000 deaths occur due to liver cancer while up to 758,000 deaths are estimated to occur due to liver cirrhosis (Seyed and Farshid 2014).
Approximately 80% of HCV infected persons will develop chronic hepatitis. Eleven percent will progress to liver cirrhosis for the period of 20-year time interval. Life-threatening will be as a result of liver failure which leads to hepatocellular carcinoma (Te andJensen 2010, Longo et al., 2011). Blood products, needle or syringe sharing among members of intravenous drug parties or undergoing a needle stick by health workers leads to transmission of HCV. Other risk factors are high-risk sexual behaviors, tattooing, reused and unsterilized dental and surgical instruments, and unsterilized laboratory equipment (Samimi-Rad et al., 2012). About 80% of HCV infected persons are asymptomatic. Infected persons with acute HCV exhibit symptom ranging from fever, joint pain and jaundice.
Asymptomatic individuals are more difficult to identify (Lozano et al., 2012: Lucas et al., 2014. Most IDUs with persistent infection are unaware of the infection, screening programs to identify patients will be required to prevent silent progression of the disease (Fabienne and Jay 2017; Mohd et al., 2013;Lavanchy, 2011). In most case HCV is often first diagnosed in late stage. Due to slow and silent onset, many patients are unaware of their infection and at least 40% cases remain undetected. Chronic hepatitis C infection is difficult to assess, because it is frequently subclinical (Te and Jensen, 2010). Patients with chronic hepatitis C are at risk of cirrhosis and hepatocellular carcinoma and their contacts (especially injecting drug users, IDUs and commercial sex workers CSWs) at risk of acquiring the infection through exposure to the virus. Given this situation, it appears that there is no clear understanding of the contextual factors that continue to fuel the upsurge in HCV infections among the key populations such as IDUs and CSWs (Kenya drug laws and human rights 2018). Molecular genotyping of HCV identified in patients is necessary, for proposing therapeutic options (Longo et al., 2011). It is assumed that some genotypes are more common in certain areas or groups of people. The high number of chronically infected individuals, the burden of disease and the absence of a vaccine indicates that treatment will form part of the control of the disease (Korir, 2013). The aim of this study was to determine the prevalence, social characteristics and HCV genotypes circulating among IDUs in rehabilitation centers in Kilifi County Kenya.

Study site and study design
This study was conducted in Kilifi County located within the Kenyan coastline. The design was cross sectional. Participants were recruited from harm reduction/rehabilitation centers within Malindi, Watamu, Kilfi town and Mtwapa {note that Kilifi town and Mtwapa is one centre splited into two called Muslim Welfare Association (MEWA)} located at Kilifi County.

Sample collection, transportation and storage
After informed consent, Socio-demographic data was captured using questionnaire which was administered at the facilities. The data captured included, name of centers, date of birth, residential area, marital status, religion, occupation and the level of education.
Further five milliliters of venous blood was collected into EDTA vacutainer tubes. They were then transported in dry ice from rehabilitation Centers to KEMRI laboratories for analysis.

Sequence analysis
Samples were aligned with the representative sequences for each major genotype and subtype selected from the HCV database and Gene Bank using the Multiple Sequence Alignment Program, ClustalW. Homology and evolutionary distance pair-wise comparisons for percent nucleotide were made. (Medhat et al., 2014). The phylogenetic analysis of HCV isolates was performed with MEGA7.0.14software.

Statistical Methods
Statistical Package for the Social Sciences (SPSS) version 20 software (SPSS Inc., Chicago, IL, USA) was used for statistical analysis. Non-parametric Mann-Whitney U test was used to compare between genotypes with respect to quantitative variables. Chi square was used to analyze association between social demographics and circulating HCV genotypes.
A p-value of less or equal to 0.05 was considered statistically significant (Valentine et al., 2018).

Demographic characteristics of study participants
A total of 127 IDUs out of 4587 registered patients were recruited for the study {107 (84.2%) were male and 20 (15.7%) were female}. The samples in each site were as follows; Malindi, (n =35), Watamu (n =43), and Kilifi and Mtwapa (n =49) respectively. The mean age for the three centers was found to be 33.7 years.

Prevalence of HCV among study participants
Anti-HCV positive included 10 IDUs from Kilifi, 4 from Watamu and 14 from Malindi making a total of 28 out of 127 participants. Twenty three IDUs (82.1%) were male while 5 (17.9%) were women. Of the 28 IDUs, 11 (8.7%) male were confirmed to be HCV positive using PCR as compared to none for female IDUs. A total 28 participants screened, seroprevalence was established at 22.1%. With the highest prevalence found in males (18.1%; n=23) and low in females at (3.9%; n=5) (P=0.004). The seroprevalence in Malindi was 40%, Watamu 9.3% and Kilifi and Mtwapa 20.4% respectively. Table 1 Serology outcome and demographic characteristics The highest number of injecting drug users was in the age group of 31-35(39%),it also had a higher number of IDUs who tested positive 8 (20.5%) as compared to other age brackets. In the study, 35 IDUs sampled were married, 68 single and 24 divorced.  Table 2 Polymerase chain reaction (PCR) and genotyping The 28 samples which were positive were subjected to PCR, 11 (39.3%) were confirmed positive (all males) and none of the 5 female IDUs was positive. The 11 samples were then subjected for sequencing and results gave 3 out of the 11 samples as HCV Genotype 1 subtype a (HCV1a) while 8 were HCV Genotype 4 subtype a (HCV4a). Table 3 Phylogenetic tree construction With reference of Full-genome consensus sequences for different HCV genotypes (including genotypes 1, 2, 3, 4, 5 and 6), there was only genotype 1 and 4 ( Figure 1). The rest there was no evident on the tree because there were no associated isolates.
Maximum composite likelihood algorithms were utilized, and phylogenetic trees were constructed by the neighbor-joining method. The reliability of different phylogenetic groupings was evaluated by using the bootstrap re-sampling test from the MEGA program (1,000 bootstrap replications). (Medhat et al., 2014;Charlotte et al., 2019).

Discussion
In this study we have managed to establish a higher prevalence of HCV in male injectors107 (84.2%) than females 20 (15.7%). We have also shown a changing trend of HCV genotype distribution with genotype 4a becoming more prevalent in this region as opposed to the traditional genotypes 1a, genotype 1a can be seen in Watamu and Malindi but not in Kilifi, it also shows that it is the first time to report HCV4a in Kilifi. Hepatitis C patients were found to be illiterate, it shows that lack of knowledge about the disease and its precautions become a strong cause for disease. In this study, there is an increase of HCV infection as compared to the study which was done by (Mwatela et al., 2015) in the entire Coast which showed a prevalence of 16.7%. We have shown that there is increased diversity along the coastal strip centers like Mtwapa which is part of Kilifi town both are under Muslim welfare association (MEWA).

Genotyping of hepatitis C virus (HCV) is considered an important tool for epidemiological
and clinical studies and valuable marker for disease progression and response to antiviral therapy (Amen et al., 2017). However, development of antiviral drugs, vaccines, and genotyping assays has a major impact on HCV this is because HCV has high levels of genetic diversity (Cuypers et al., 2015;Medhat et al., 2014;Vanessa et al., 2018). In this study, we have confirmed that only two genotypes circulating in Kilifi County; this can form a basis for more targeted control for the 2 subtypes in the region. Hepatitis C Virus diversity is very essential in response to antiviral therapy (Medhat et al., 2014;Tawhida et al., 2015). More severe liver disease and more elevated liver enzymes have been highly associated with HCV genotype 1 (Chakravarti et al., 2011). The endemicity of HCV genotypes in some areas is revered with multiplicity and diversity of subtypes and strains. A good example is origin of genotypes 1 and 2 to West Africa and genotype 4 to Central Africa (James et al., 2014). This can be done by using a phylogenetic analysis to revealed two monophyletic clusters (bootstrap value,∼87) containing HCV infected patients in Kilifi County from which a partial 5′UTR sequence was available.
The largest cluster contained 87% of HCV sequences were identified as genotype 4, with isolates M01, M02, M05, M07, M23, K20, W10, and W14. This isolates clusters with isolates from: Portugal, France, Egypt, Cyprus, Southeast Asia, Middle East and Saudi Arabia. On the other hand genotype1a Isolates were K08, K14 and W17 Clusters with isolates from: America, Japan, Indonesia, China and India. Genotype diversity is particularly high in China and many Southeast Asian countries and also in Western Europe and Australia, perhaps as a result of population immigration from Africa and/or Asia (International migration data 2012). In countries like these, vaccine efficacy at a population level would be dependent on the generation of cross reactive immunity; an alternative approach could also be the development of vaccines hosting different immunogens. In this study, HCV genotype 4a and genotype1a is predominant among the population of drug users, this suggest that there is a need for continuous monitoring of these for better treatment outcomes that entirely depend on HCV genotyping results. With global travel, increasing of tourist to Kilifi Count and the geographic zone of this region dictates the distribution of this genotypes in this region (Rong et al., 2014).
Due to lack of vaccine and effective therapy, the prevention of HCV infection has been a great challenge to developing country and owns one-fifths of the world's population (Tanaka et al., 2011). World Health Organization Assembly approved on 2016 the Global Health Sector Strategy to eliminate hepatitis infection by 2030, (Assembly WHOS-NWH 2016). It introduced global targets for the care and management of HCV including 90% reduction in new cases of chronic hepatitis C, 65% reduction in hepatitis C deaths, and treatment of 80% of eligible people with chronic hepatitis C infections (WHO 2016). To achieve these goals, the country need to develop national policies based on reliable epidemiological evidence (Saraswat et al., 2015). In Kilifi County, Screening populations at risk and Counseling program to IDUs with history of sexual contact or sharing of items should be done to stop spread of HCV in the region. Tourists born in countries where HCV prevalence is high are supposed to be screened before accessing entry to Kilifi County.
However, data are often outdated and conflicting, making evidence-based policy and resource allocation difficult.

Conclusions
In this study, the prevalence of HCV in the area seems to be increasing as compared to previous studies. It is also the first time to report HCV4a in Kilifi town as compared to    Phylogenetic neighbor-joining trees of Hepatitis C Virus (HCV) 5′UTR partial sequences in Kilifi County, it revealed two monophyletic clusters (bootstrap value,∼87) containing Hepatitis C Virus Genotype one Subtype a (HCV1a) and Hepatitis C Virus Genotype four Subtype a (HCV4a) among infected patients in the County.