Immunoassay of anti-HCV and seroepidemiological surveillance of hepatitis C virus infection

Citation :Egypt.Acad.J.Biolog.Sci. ( C.physiology and Molecular biology ) Vol.7(2)pp1-9(2015) Egyptian Academic Journal of Biological Sciences is the official English language journal of the Egyptian Society for Biological Sciences, Department of Entomology, Faculty of Sciences Ain Shams University . Physiology & molecular biology journal is one of the series issued twice by the Egyptian Academic Journal of Biological Sciences, and is devoted to publication of original papers that elucidateimportant biological, chemical, or physical mechanisms of broad physiological significance. www.eajbs.eg.net Provided for non-commercial research and education use.

These genotypes can differ by up to 50% of their nucleotide sequences, and the virus has a high propensity to mutate.In a large population-based study, 1.8% of a large household-based sample was positive for anti-HCV antibody (2.3% in adults 20 years or older), which would translate into an estimated 3.9 million infected persons in the U.S (Alter et al., 1999).74% of them had viremia, indicating chronic infection (an estimated 2.7 million).Studies have reported a higher prevalence of anti-HCV antibodies, with the highest rates consistently in intravenous drug users (Alter,1997 andBell et al.,1990).The incidence of HCV infection has fallen since the 1990s.The yearly incidence of HCV infection was estimated to average 230,000 cases per year in the 1980s, but by 2001 had declined to 25,000 cases per year.
In a French population-based study, 24% (17/72) were aware that they were seropositive for HCV infection (Dubois et al.,1997).HCV infection is a leading cause of complications from chronic liver disease in the United States.HCV-related end-stage liver disease is now the most common indication for liver transplantation among American adults, accounting for over 30% of cases, and there was a 5-fold increase in the number of patients with HCV who underwent liver transplantation between 1990transplantation between and 2000transplantation between (Busch, 2001;;Kim, 2002;Seeff et al.,1992;Seeff et al.,1994).Chronic HCV infection can also cause morbidity in the absence of cirrhosis or other serious complications.HCV infection without cirrhosis is associated with significantly worse quality of life measures and symptoms (primarily fatigue) compared to the general population, though confounding factors such as intravenous drug use and other co-morbid conditions have not been wellcontrolled in studies (Kenny-Walsh,1999;Koff, 1999 andFoster et al.,1998).One study in women with a low prevalence of intravenous drug use, however, found high levels of psychological distress and impaired quality of life in women with anti-HCV antibodies that did not correlate to the presence or absence of chronic infection (Coughlan et al., 2002).Hepatitis C virus (HCV) has ability to persist in most immunocompetent adults.Although HCV activates an innate immune response, it employs an elaborate set of mechanisms to evade interferon (IFN)-based antiviral immunity.By comparing innate and adaptive immune responses to HCV with those to hepatitis A and B viruses, prolonged innate immune activation by HCV impairs the development of successful adaptive immune responses (Su and Barbara,2014).Hepatitis C virus have the capacity to generate a strong cellular immune response against the virus and avoid persistent infection, and perhaps do so repeatedly after reexposure (Lisa et al.,2006).HCV-specific antibodies generally develop 2-8 weeks after exposure in humans and remain throughout the course of chronic infection.Antibody levels are generally low, with limited affinity maturation or isotype switching (Chen et al.,1999).In those who clear the virus, antibody levels gradually diminish in titer, with approximately 50% of individuals having no detectable antibodies by 20 years after primary infection (Takaki et al., 2000 andVillano et al., 1999).Although this humoral immunity may be part of an effective immune response in the minority of individuals who clear the primary infection, antibody generation is not an absolute requirement for viral clearance (Abe et al.,1992;Farci et al.,1996;Mehta et al., 2002).Studies have shown that individuals with hypogammaglobulinaemia clear the virus in proportions similar to the general population (Bjoro et al.,1994;Razi et al.,2001 andChapel et al., 2001), and recovery does not correlate to anti-HCV antibody titers or to the levels of antibodies directed to the envelope glycoprotein's, E1 and E2 (Bassett et al., 1998;Kobayash et al.,1997 andGrellier et al.,1997), although it should be noted that these studies did not assess neutralizing antibodies.

Study setting and design
This study was carried out over a Five-month period at a Maternity hospital and King Khalid hospital in Hail region, Saudi Arabia.

Participants:
A total of 100 participants in the agegroup of 18-63 years, attending the clinic at Maternity and King Khalid hospital, Hail, Saudi Arabia,between October 2013 to February 2014 were studied.The approval of the institute's was obtained priorto the sample collection; informed writtenconsent was obtained from all the participants.Detailed clinical historyand conventional laboratory investigations wereconducted.

Questionnaire:
A preformatted questionnaire including the demographic data, socioeconomic status and medical history wascompleted during patients followup.The questionnaires included questions on Age, gender, Family income, occupation and educational level and patient history.

Statistical analysis:
Statistical analysis was performed by using SPSS for Windows version 12.0 (SPSS INC., Chicago, III., USA).Data was presented as mean ± SD.Chi-square analysis µ2 was used in findings on comparison of HCV infection positivity according to individual characteristics.Evaluation was carried out at the 95-99% confidence interval and P< 0.05 was considered statistically significant.

Serum samples:
Three mL of venous blood were collected from 37 participants, in a container with strict aseptic precautions.The serum was used for serological evaluation of ani-HCV infection.

ELISA kits for detection of anti-HCV(Monolisa™ Anti-HCVPLUSV ersion 2)
An indirect immunoenzymatic technique allowing the detection of the antibodies associated with an infection by Hepatitis C virus in patient serum.Monolisa™ Anti-HCV PLUS Version 2 is based upon the use of a solid phase prepared with purified antigens, ٣recombinant proteins produced by E.coli from clones selected in the non structural area (NS3 and NS4) andin the structural area of the hepatitis C virus genome.

RESULT I-Incidence of HCV in Hail region February to December 2013
During February to December 2013, (165 blood donors) were enrolled in this study,79% out of them are positive for HCV Assay, As show in

II-Seroprevalence of TORCH in Hail region
Among the participants, HCV positivity percentage was found to be (1%) (One /100) in our study.It was found that, the average age of the patient was (18 to 63 years old), the majority came from Hail city, as shown in Table (1).

Comparison of Knowledgement about HCV infection and HCV positivity
Obtained data revealed that, only 1% of participants know about HCV and methods of infection, as shown in Table (2).

Comparison of Knowledgement about symptoms of HCV infection
Among the participants, only 1% of them know about symptoms of chronic phase of HCV infection, as shown in Table (3).

Comparison of Knowledgement about serological and molecular tests for detection of HCV infection:
Obtained results illustrated that, only 1% of participants Know and conduct serological test for detection of HCV, as shown in Table (4).

Detection of anti-HCV in patients serum by using ELISA Kits:
Thirty seven serum samples were examined for anti-HCV, thirty of them were positive withtiter ranged from (10.512 to 1859.395IU/ml), while seven samples were negative, as shown in Table (5) and Fig

DISCUSSION
Incidence of HCV infection blood donors in Hail region was 79%.Hepatitis C virus prevalence rates are estimated to range between 5.5% in Africa, 4.6% in the Eastern Mediterranean region, 4% in the Western Pacific region, 2% in South East Asia, 1.7% in the United States of America (USA), and 1% in Europe (Shobokshi et al.,2003).There has been very high prevalence rate of HCV reported in Egypt in the past 28% (Sy and Mazen, 2006).The study showed 1.3% seroprevalence of HCV Ab among general population in central region of Yemen (Ibb city).The frequency of HCV Ab seropositivity was found to be higher than that reported in southern part of Yemen (Aden city) 0.6% and lower than northern part of Yemen (Sana'a city) 2.3% and southern islands (Socotra island) 5.1% ( Bajubair et al., 2008).Such differences in prevalence rates may be explained by differences in health resources and educational levels in different regions in Yemen or due to methodological differences between studies.(Mahaba et al. 1999)  The seroprevalence of HCV positivity in Hail region was 1%, with average age of patient (18 to 63 years old) and these result was agreed with (Mahdi et al., 2000 andMadani, 2007) who mentioned that, prevalence assessed from Saudi blood donor screening centers indicates HCV infection rates of 0.4-1.1%.Declines in HCV prevalence rates were also noted in the blood bank database of King Khalid University Hospital in Riyadh, from 0.58% in 1996 to 0.08% in 2006.A summary report compiled by the WHO mentions 437,292 official reports of HCV infections among persons living in the KSA, giving an estimated prevalence of about 1.8% (WHO,2009).
The obtained data revealed that titer of anti-HCV in patient's serum was ranged from (10.512 to 1859.395IU/ml).(Khayriyyah et al., 2013) mentioned that the number of persons with anti-HCV in the world has increased from an estimated 122 million (P: 2.3%, 95% UI: 2.1%-2.5%) in 1990 to an estimated 184 million (P: 2.8%, 95% UI: 2.6%-3.1%) in 2005.However, given the cross-sectional nature of prevalence data, this global rise in prevalence and changes observed in East Asia, Western Europe, and West sub-Saharan Africa may reflect changes in compositional data or global shifts in age patterns rather than changes in disease epidemiology.

CONCLUSION
In conclusion, the results of the present study hint that, seroprevalence of HCV positivity in Hail region was 1%, the relatively low prevalence of HCV in our study group may well be due to an improvement in the control the testing for infectious diseases in the home countries of the expatriates.

Figure ( 1 ):
Figure (1): Incidence of HCV in Hail region February to December 2013.II-Seroprevalence of TORCH in Hail regionAmong the participants, HCV positivity percentage was found to be . (2).

Table 1 :
Comparison of demographic characteristics and HCV positivity.

Table 2 :
Comparison of Knowledgement about HCV infection and HCV positivity.

Table 3 :
Knowledgement about symptoms of HCV infection.

Table 4 :
Knowledgement about serological and molecular tests for detection of HCV infection.