PREVALENCE AND GENOTYPING OF HEPATITIS C VIRUS IN HEMODIALYSIS PATIENTS AND EVALUATION OF HCV-CORE ANTIGEN TEST IN SCREENING PATIENTS FOR DIALYSIS IN SANA'A CITY, YEMEN

Hepatitis C virus infection is a constant worldwide public health concern. The prevalence of HCV infection is higher in patients on chronic haemodialysis (HD) than in the general population. Despite the control of blood products, hepatitis C virus transmission is still being observed among patients undergoing dialysis. Detection systems for serum HCV antibodies are insensitive in the acute phase because of the long serological window. Direct detection of HCV depends on PCR test but this test is not suitable for routine screening. The objective of this study was to determine prevalence of HCV, genotyping and if HCV core antigen test could be a better alternative to NAT techniques for the diagnosis of HCV infection during the window period and whether the sensitivity for antibody detection is preserved. We screened 159 patients on long-term dialysis by HVC antibodies test, PCR HCV-RNA and HCV core antigen test by commercial tests. The prevalence of HCV was 10.7% (17 patients) and genotype 4 was the most common one (64.7%). The sensitivity of HCV core antigen test was 94.1%, the specificity 100%, the positive predictive power 100%, and the negative predictive power 97.9%. In conclusions; patients on maintenance HD in Yemen have a high prevalence of HCV infection comparing with general population; and genotype 4 is predominant. The performance of serological detection of HCV core antigen was better than that of HCV antibodies test and may be an alternative to nucleic acid amplification technology (NAT) for routine monitoring of patients on chronic dialysis.


Introduction
An estimated 143 million people (2%) worldwide are infected with hepatitis C as reported in for 2015. 1 In 2013 about 11 million new cases occurred. 1 It occurs most commonly in Africa and Central and East Asia. 2 About 167,000 deaths due to liver cancer and 326,000 deaths due to cirrhosis occurred in 2015 due to hepatitis C. 2 In Yemen HCV antibodies among general community showed a steady decline to less than 0.5% 3,4 , however among HCV risk groups such as dental workers and public health center workers, it was were 5.5% and 11.5% respectively. 5,6 Limited information is available among in dialysis patients for dialysis in Yemen. HCV infection is more common among in dialysis patients for dialysis than in healthy populations worldwide. The Dialysis Outcomes and Practice Patterns Study (DOPPS) reported an in a general prevalence of 13.5 percent among adult hemodialysis patients randomly selected from 308 dialysis services in developed countries (France, Germany, Italy, Japan, Spain, the United Kingdom, and the United States). 7 A study from Australia and New Zealand 8 in HD patients (n = 23,046) reported an independent and significant association between anti-HCV positive serologic status and all-cause mortality over a 10-year follow up (HR, 1.25, 95% CI 1.07-1.46, P = 0.004). Regardless of the control of blood products, HCV transmission is stationary being observed among HD patients. HCV infection diagnosis is usually diagnosed rooted in the detection of an anti-HCV antibody, while it goes undetected in the first 4-6 weeks of infection (so-called window period). Furthermore, patients positive for anti-HCV antibody include both those who are actively infected and those who have recovered from infection. 9 Kidney Disease Improving Global Outcomes (KDIGO) clinical practice guidelines for the prevention, diagnosis, evaluation, and treatment of hepatitis C in chronic kidney disease 10 recommended the use of nucleic acid amplification technology (NAT). A quantitative HCV core antigen (HCVcAg) test has been developed for the confirmation of viremia in patients with hepatitis C. This test can detect total nucleo-capsid core antigen whose sequence is highly conserved across HCV genotypes. A number of studies in the general population have highlighted the importance of HCV core antigen detection as an alternative to NAT for early diagnosis of infection, as direct marker of viral replication in chronic phase of infection and as relevant marker for predicting and monitoring the response to therapy. 11 Few studies exist about the efficacy of HCV core antigen test in patients on chronic HD in the early diagnosis of HCV infection. [12][13][14] The objective of this study was to determine prevalence of HCV, genotyping and if HCV core antigen test could be better an alternative to NAT techniques for the diagnosis of HCV infection during the window period and whether the sensitivity for antibody detection is preserved.

Subjects and Methods
The study was performed in the haemodialysis units of Al-Thorah hospital haemodialysis unit. A total of 159 patients were enrolled in this cross sectional study in 2016; patients gave informed consent and thus the whole patient population were investigated. All patients underwent chronic haemodialysis treatment for end stage renal disease during the study period. The laboratory tests works were conducted in Al-Awalagy Medical laboratory. Anti-HCV antibody was measured by a third generation commercial ELISA (Enzymun-Test Anti-HCV; Boehringer Mannheim, Germany). The third generation assay detects antibodies for three viral antigens (c22-3, c200, and NS5). HCV-RNA and viral genotype were assessed using by PCR (TaqMan Roche). All tests were carried out and interpreted strictly in accordance with the manufacturer's instructions.

Statistical Analysis
The performance of the HCV core antigens and antibodies HCV test were done by comparing to HCV-RNA PCR test. The following parameters were calculated: sensitivity%, Specificity%, false positive% (FP), false negative% (FN), positive predictive value (PPV); and negative predictive value (NPV). To gGender and age group's which are possible associated risk factors for HCV infection, were assessed. tThe data were examined in a case-control study format. For HCV, persons with evidence of previous or current infection with HCV were matched up with those who were HCV negative.

Ethical Consideration
Ethical clearance for the study was taken from the Faculty of Medicine and Health Sciences Research Review Committee. Informed Consent was taken from the volunteers before the collecting specimens and file questionnaire. Table 1 shows the prevalence rate of HCV ribonucleic acid (RNA), and associated odds ratio for different sex and age of a sample of hemodialysis patients in Sana'a city. The crude prevalence rate of HCV among HD patients was 10.7%, for male patients was 14.3%, higher than 8.7% for of female patients. When age was considered, there was an increasing rise trend of HCV infection with increasing age. The genotype distribution in 17 HCV-positive patients is shown in Table 2. Overall, HCV genotype 4 was the most predominant genotype (64.7%) followed by genotype 1a and 1b (29.4%) and 2a (5.9%). Table 3 shows the prevalence rate of different HCV markers in 159 haemodialysis patients in Sana'a city. A Seventeen (17) HD patients were HCV ribonucleic acid (RNA) positive, 14 HD patients were HCV antibodies positive and 16 HD patients were HCV core proteins positive. Table 4 shows the performance of HCV antibodies test and HCV Core protein test compared ing to HCV ribonucleic acid (RNA) among haemodialysis patients in Sana'a city. The sensitivity of HCV core antigen test was 94.1%, the specificity 100%, the positive predictive power 100%, the negative predictive power 97.9%, false positive rate 0.0% and false negative rate 5.9%. However less reliable results were found for HCV antibodies test in which the sensitivity of HCV antibodies test was 70.6%, the specificity 98.9%, the positive predictive power 100%, the negative predictive power 97.9% false positive rate 1.41% and false negative rate 29.4%. Thus 1.41% (FP) of the entire study population was HCV positive but could not be diagnosed by routine HCV antibody testing, while 0% (FP) of the entire study population was HCV positive could not be diagnosed by routine HCV core antigen test.

Discussion
Information on of the prevalence of HCV in the general population and in the various high risk groups such as HD patients, prevalence of genotypes and evolution of tests used for screening HCV are important in the prevention and control HBV infections. Unfortunately, there is little information available on these topics, particularly from Middle East countries and more specifically from Yemen. HCV infection continues to be the most frequently recognized cause of liver damage in patients with CKD. 15 Although a severe clinical course of HCV-related liver disease seems unusual in most HD patients and cirrhosis is an infrequent event among dialysis patients, longitudinal studies have found an independent and significant relationship between anti-HCV antibody positivity and reduced patient survival. 16,17 The Dialysis Outcomes and Practice Patterns Study (DOPPS) on HD patients in three continents [18] had reported an independent and significant association between positive anti-HCV antibody and mortality risk (adjusted relative risk, 1.17; P < 0.0159). Fabrizi et al. 19 showed that HCV-seropositive HD patients had higher rates of liver disease-related death than their sero-negative matching parts, but that cardiovascular and infectious disease related mortality rates were similar. 19 Ohsawa et al. showed that seropositivity for anti-HCVcAg is independently associated with increased all-cause, cardiovascular, and liver disease related mortality in HD patients. 20 In the current study in Yemen the crude prevalence rate of HCV among HD patients was 10.7%, (male rate=14.3%, female rate= 8.7%). This result is indicative ed that HCV infection is more common in dialysis patients in Yemen than in healthy populations (0.5-5%). [3][4][5] The current result also is slightly lower than the Dialysis Outcomes and Practice Patterns Study (DOPPS) which it reported an overall prevalence of 13.5% among adult hemodialysis patients randomly selected from 308 dialysis facilities in developed countries. 7 In addition, our study results show that HCV genotype 4 is the predominant genotype (64.7%) among Yemeni patients followed by 1a and 1b (29.4%) and 2a (5.9%). This data is similar to that reported in Middle East countries, and previously in the Yemen; where genotype 4 is the predominant. 4,[21][22][23][24] It is important to diagnose a hepatitis C virus infection in the acute phase in order to reduce the incidence of this infection in high-risk populations like HD patients. 25 Biochemical evaluation of HCV infection in patients with CKD is inaccurate. Serum aminotransferase values are typically lower in dialysis patients than the non-uremic populations. 26 Thus we carried out this study to evaluated the performance of HCV antibodies test and HCV Core protein test comparing to HCV ribonucleic acid (RNA) among our haemodialysis patients (table 4). The sensitivity of HCV core antigen test was 94.1%, the specificity 100%, the positive predictive power 100%, the negative predictive power 97.9%, false positive rate 0.0% and false negative rate 5.9%. However less reliable results were found for HCV antibodies test in which the sensitivity of HCV antibodies test was 70.6%, the specificity 98.9%, the positive predictive power 100%, the negative predictive power 97.9% false positive rate 1.41% and false negative rate 29.4%. Our results is similar to that reported previously in which detection systems for serum HCV antibodies are insensitive in the acute phase because of the long serological window. 7 Also, the direct detection of HCV depends on NAT techniques with several problems: frequent unavailability, considerable skill requirement, limited reproducibility, and overall important costs. HCV detection by PCR-RNA, although widely accepted as a gold standard test in the diagnosis of HCV infection in CKD patients, it is not suitable for routine screening. 7 Thus from our results, HCV core antigen quantification assay has is proved useful for an early diagnosis of HCV infection in community-based and in dialysis populations. Also, HCV core antigen may be an alternative to HCV-RNA detection, since no subjects, who were negative for HCV core antigen, were positive for HCV-RNA (false negative=0.0%). Our result is similar to that reported in a large population-based cohort studies by Ohsawa et al. 27 ; and by Kato et al. 28 in which no subjects, who were negative for HCV core antigen, were positive for HCV-RNA; also Ohsawa et al. 27 and Kato et al. 28 suggests that detection of HCV core antigen combined with anti-HCV antibody is useful in predicting long-term survival prognosis of persistent HCV infection in HD patients.
Finally, from our experience HCV core antigen test is both a cost-effective (a single sample has a 40$ charge for PCR HCVRNA and a 5$ charge for HCV core antigen test) and a less labour-intensive alternative to NAT tests. These features make it a routine this assay useful for routine of chronic dialysis treatment patients.

Conclusion
Patients on maintenance HD in Yemen have a high incidence and prevalence of HCV infection and genotype 4 is the predominant one. Serological detection of HCV core antigen may be an alternative to NAT techniques for routine monitoring of patients on chronic dialysis towards the prevention of HCV spread. HCV core antigen is an accurate marker for early identification of HCV infection; it can improve virological monitoring and integrate the diagnosis of acute hepatitis C in dialysis population. The minimal cost and its easiness make this assay useful for routine longterm dialysis treatment patients. Furthermore, screening for HCV antibodies alone does not exclude infection with HCV.   Sensitivity: Probability that test will be positive, given the patient is disease. Specify: Probability that the test will be negative, given the patient is disease free. FP=false positive : Probability that test will be positive, known the patient is disease free. FN= false negative: Probability that test will be negative, known the patient is diseased. PPV= positive predictive value: Probability that a patient is diseased, given a positive test. NPV= negative predictive value: Probability that a patient disease free, given a negative test.