INTRODUCTION:

Hepatitis is term to describe a nonspecific liver inflammation. Liver is the largest and most complex internal organ of human body.

It performs wide range of functions ranging from detoxification of hormonal substances to synthesis of various products. Naturally liver is vulnerable to a wide variety of insults- metabolic, inflammatory, toxic, microbial, circulatory and neoplastic. Among the patients suffering from liver diseases, viral hepatitis is a common clinical condition.¹

Viral hepatitis has been a part of recorded history since the time of Hippocrates (Bockus Gastroentrology, 5^th Edition) for centuries, epidemics of hepatitis have devastated military and civilian communities throughout the world². Initially transmission of disease was attributed to non-parenteral routes, such as person to person contact or exposure to contaminated food or water. This was known as epidemic (infectious) hepatitis.³

However in 1885, in Bremen, Germany significant event occurred. Nearly 200 shipyard workers become Jaundiced several months after vaccination against small pox with human lymph. Also there was no jaundice recorded in unvaccinated persons. This suggested another mode of transmission of disease with long incubation period and by parenteral route (Lurman 1885).⁴

Over the next century viral hepatitis was classified into two diagnostic categories- one infectious hepatitis and another serum hepatitis. Later on the names ‘Hepatitis A’ and ‘hepatitis B’ for serum hepatitis were proposed (Mac Callum, 1947)⁵.

No further progress in the knowledge of viral hepatitis was made until 1965, when Blumberg in Philadelphia, discovered an unusual antibody in the serum of hemophilics, who had received multiple blood transfusions. This antibody reacted with sera of an Australian aborigine but not with variety of sera from other people .Blumberg et al, therefore named this antigen present in sera of Australian aborigine as ‘Australia antigen’ This antigen is now known as the Hepatitis B surface antigen (HbsAg).⁶ Years later in 1973, Feinstone et al discovered hepatitis A virus, the agent responsible for infectious (epidemic) hepatitis.⁷

The majority of cases of hepatitis until 1975 were attributed to either HAV or HBV. But still there were some cases of hepatitis particularly post transfusion hepatitis, which could not be accounted for by either of these viruses. And to explain these cases name of “non A, non B hepatitis (NANBH) was proposed by Feinstone in 1975⁸.

In 1977 another breakthrough occurred when Rizzetto identified another virus responsible for viral hepatitis and named it as Delta agent (Hepatitis D virus). This virus was an incomplete virus because it was responsible for transmission of the blood borne type of hepatitis in combination with HBV only and not individually.⁹

Initially the diagnosis of Non-A, Non-B hepatitis was made by excluding HAV and HBV infections. This led to the search of causative agent of NANBH. Various studies revealed that NANB agent is not a single virus but different viruses responsible for different types of

NANBH.

These cases were then identified by Balayan¹⁰ (in Russia, 1983) and Houghton¹¹(in USA,1988) as to be caused by two different viruses ,HEV and HCV respectively. Balayan isolated hepatitis E virus responsible for water-borne NANB hepatitis and Houghton isolated hepatitis C virus causing blood-borne NANB hepatitis. Later in 1989, Choo et al cloned the hepatitis C virus.¹²

The study has been undertaken to find out the prevalence of hepatitis C virus infection in the blood donors attending the blood bank of Gandhi Memorial and Sanjay Gandhi Memorial Hospital, Rewa associated with department of Pathology Shyam Shah Medical College Rewa (M.P.).

MATERIAL AND METHOD:

This study was carried out to assess the prevalence of HCV infection among the blood donors, who are attending blood bank for blood donation; by detecting antibodies to HCV in their blood firstly by rapid kits and confirmed by ELISA method. The blood donors (voluntary/replacement) attending the blood bank of Sanjay Gandhi Memorial Hospital, Rewa were selected. This study was carried out from September 2013 to June 2014 in the department of Pathology, S.S. Medical College, Rewa (M.P.).

In our study 5281 samples were collected and screened for the presence of antibodies to HCV by rapid chromatographic immunoassay test strips (IND Diagnostic Inc. 1629 Fosters Way, Delta B.C. Canada V3M6S7). The reactive samples are further confirmed by Enzyme Linked Immunosorbent assay (Innova HCV Elisa Kit, Span Diagnostics Limited, Surat, Gujarat, India).

Methodology of HCV One Step Rapid Kit Test (according to manufacturer’s instruction):

It is a direct binding screening test for the detection of antibodies to Hepatitis C Viruses, which causes chronic liver diseases. Recombinant HCV (NS3, NS4 and NS5) and structural core proteins antigens are employed to specifically identify HCV antibodies specifically.

Principle:

Rapid kits test HCV is membrane based test, which is coated with recombinant antigen (NS3, NS4, NS5 and core) specific to HCV antibodies at test region “T”. The control region “C” is coated with protein A. Protein A Colloidal gold conjugate is used as tracer. When the test sample most further on the membrane assembly, the Gold Conjugated Protein A complexes with the HCV antibodies in the serum sample. This complex moves further on the membrane will be trapped by the HCV combined antigens coated on the test region “T” results in the formation of pink/purple colour line on the test region which confirms the presence of HCV antibodies in the sample, absence of this band on test region indicates absence of antibodies specific to HCV. The unreacted conjugate moves further on the membrane, ends up in forming coloured control line. The control line serves to validate the test result. No band at region “C” indicates the test is “Invalid”.

Methodology of Microwell ELISA Method for HCV:

ELISA is an in vitro method for the qualitative detection of antibody to hepatitis C virus in human serum or plasma. It is a third generation diagnostic kit detecting antibody to hepatitis C virus. The kit is composed of 3 kinds of fusion protein KHCV Ub – core 518 (core /NS3 fusion protein), KHCV Ub – E1E2NS4 (E1E1/NS4 fusion protein), KHCV ub – NS5. These antigens are produced from recombinant protein in E.coli and yeast.

Principle:

In this test specimen is diluted 10 fold in a specimen diluent. HCV antibodies in a test specimen are incubated with recombinant HCV antigens core 518, E1E2/NS4, NS5 bound on microwell (1^st reaction). After washing, HCV antibodies in a specimen bound to antigen on microwell are detected by second antibody – HRP (anti human IgG conjugated with peroxidase). This step is 2^nd reaction. After washing, the following step is substrate color development reaction. That is, o – phenylenediamine (OPD) solution containing hydrogen peroxide is added to the wells. If bound second antibody – HPR is present, the OPD will be oxidized resulting in orange – yellow color development dependent upon the amount of bound second HRP. This is proportional to the amount of anti- HCV present in the test specimen. The substrate reaction is stopped by the addition of 4N sulfuric acid. The color intensity is measured with an ELISA reader (spectrophotometer) at 492nm.

ETHICAL CLEARENCE:

Ethical clearance was taken by institutional ethical committee Shyam Shah Medical College Rewa (M.P.). Written consent was taken by all patients and donors, involved in the study.

STATISTICAL ANALYSIS

Statistical analysis was done by using Minitab 17 Pennsylvania, USA.

For non parametric data chi – square test was done. P-value <0.05 has taken as significant for all analytical studies.

RESULTS:

Table – 1-Prevalence of HCV according to type of donor

Type Of Donor	No.	HCV Positive Cases	Prevalence %
Voluntary Donor	4431	80	1.8
Replacement Donor	850	8	0.94
Total	5281	88	1.6

Statistical Inference for HCV: Chi-square test between voluntary and replacement donor X² = 3.7 and p = 0.053 (Difference is Insignificant since >0.05)

Above table shows the prevalence rate of HCV infection in voluntary and replacement donors. The overall prevalence of HCV is 1.6% (p> 0.053). The voluntary donors accounting 1.8 % prevalence and replacement donors 0.94%. The prevalence rate of HCV is higher in voluntary donors (1.6%).

Table – 2-Prevalence of HCV Positive cases according to gender

Gender	No.	HCV Positive Cases	Prevalence %
Male	4623	86	1.8
Female	658	2	0.30
Total	5281	88	1.6

Above table shows overall prevalence rate of HCV infection in male and female was 1.6%. The prevalence rate of HCV in Male and Female was 1.8 and 0.30 % respectively. The prevalence rate of HCV is higher in males.

Table -3-Prevalence of HCV in Age Range Of Donors.

Age Groups	HCV Positive Cases	Prevalence %	Male	Female
18-28	18	20.4%	18	0
29-38	37	42.0%	36	1
39-48	26	29.5%	26	0
49-58	7	7.9%	6	1
Total	88	100%	86	2

Table-3 shows prevalence of various age groups in donors. The prevalence of HCV in age group range between 18-28 years was 20.4%, 29-38 years was 42.0%, 39-48 years was 29.5% and 49-58 years was 7.9%. The prevalence of HCV was higher in age group 29-38 years it was 42.0%.

Table – 4-Prevalence Of HCV according To Occupation

Occupation	No.	HCV Positive Cases	Prevalence %
Working	1940	40	2.06
Student	1673	38	2.2
Farmer	912	1	0.1
Others	756	9	1.19
Total	5281	88	1.6

Table-4 shows the prevalence of HCV in donors based on their occupation. The prevalence of HCV in working population was 2.06%,in student it was 2.2%,in farmer it was 0.1%, and in others ( laborer, house- wives etc.) it was 1.19%. Higher prevalence was found in student group.

In our study it was also found that the prevalence of HCV in Hindus was 1.69 % and in Muslim it was 1.2% where as in others it was 0%.

DISCUSSION:

The present study was done on 5281 healthy blood donors attending the blood bank Sanjay Gandhi Memorial Hospital, Rewa from September 2013 to June 2014 were selected. First they screened with rapid card test; donors who show reactivity are further tested with ELISA 3^rd generation. The overall prevalence of HCV infection was 1.6 %.

In India, various studies have been done to determine prevalence of HCV with other blood borne diseases. On blood bank healthy blood donors studies; the overall prevalence of HCV ranges between 0.15% to 3.6%. Thus, prevalence observed in our study was within the range of national prevalence rate.

Results obtained by various workers in India have been shown in the tables along with results of the present study

INDIAN STUDIES (HCV PREVALENCE RATE IN BLOOD DONORS)

S.No.	Author	Year	Place	No. of cases	Prevalence rate (%)
1	Riddhi Jaiswal et al¹³	1997-2005	GSVM Medical college Kanpur	1,15,073	0.4
2	R.N. Makroo et al¹⁴	2001-11	Indraprastha Apollo Hospitals, New Delhi	2,06,022	0.39
3	Dharmesh Chandra et al¹⁵	2009-13	Gajra Raja ,Medical College, Gwalior	67,132	0.24
4	Yedlapati Bhawani et al¹⁶	2004-09	KIMS HOSPITAL(A.P.)	8,097	0.84
5	Beenu Thakral et al¹⁷	2001-02	PGIMER, Chandigarh	16,250	0.44
6	Sastry Jayagowri M et al¹⁸	2008-13	Charitable hospital blood bank in Pune	13,078	0.41
7	Farhan Asif Siddiqui¹⁹	2001-07	Jawaharlal Nehru Medical College Hospital, A.M.U., Aligarh	68,173	0.07
8	Dr. Anu Gupta²⁰	2004	S.S. Medical College, Rewa	1200	3.6
9	Nilima Sawke et al²¹	2006-08	People’s College Of Medical Sciences and Research Centre Bhopal	5008	0.57
10	Dr. Nirali Shah²²	2006 - 13	Sheth V.S. General Hospital , Ahmedabad.	92,778	0.101
11	Present study	2013-14	S.S. Medical College and Sanjay Gandhi Memorial Hospital, Rewa	5281	1.6

In present study prevalence of HCV is within the range (1.6%) of prevalence rates of various Indian studies on blood donors it is (0.07 – 3.6%). Results are not very close to any study it is in between the two studies Yadlapati Bhawani¹⁶ et al it is 0.84% (68/8097) at KIMS Hospital Amalapuram (AP) and Dr. Anu Gupta²⁰ studied on healthy blood donors at Sanjay Gandhi Memorial Hospital and S.S. Medical College, Rewa she made 3.6% prevalence (44/1200) which is quite higher than my study.

Similarly HBsAg various factors which need to be considered to explain the prevalence of HCV with comparison with other Indian studies. The present study includes all the donors including replacement donors (16.09%) and voluntary donors (83.9%).

Second factor is the effect of study design especially the size of the sample population, type of the donor (Voluntary/ Replacement) and efficacy of the assay used.

Riddhi Jaiswal¹³ et al studied for nine year from January 1997 to December 2005 screened with ELISA kit on 1,15,073 blood donors and found overall prevalence of 0.4% and found prevalence between male and female 1.64 and 1.57% respectively; but in present study it was 3.0 and 1.2 respectively. In present study it was higher because of different epidemiological region.

In our study maximum donors presented in the age group 29-38 year (42.0%) for HCV prevalence.

Similar findings was found by Fathi Abed A l-Gani²³ in 2011 in Jordan found prevalence of HCV 33.3% also in elder group > 50 years.

The cause of high prevalence in younger age in my study:

1) Low education level - This region is not having 100% literacy so not all population properly know about these infections and its complications.

3) Un- hygienic habit– people using un-sterilized syringing, intra venous drug users because of more frequently used.

4) No vaccination by government to prevent it (not included in national vaccination programs)

6) Lack of proper screening facility before blood transfusion so that professional donors not excluded by screening.

In our study we found that prevalence is more in student group in case of HCV (2.2%).

Causes for high prevalence in employed and student group:

1) Lack of proper awareness towards blood borne diseases.

2) Use of intra venous drugs by employed population and students. It’s emerging fashion among these group.

3) Lack of motivational programs to discourage use of narcotic drugs.

4) There are no strict punishment for drug suppliers.

5) Lack of use of condoms and other protective measures during sex.

6) Higher prostitution practice in Rewa region.

In our study we found that prevalence was more in hindu population 1.69% (and 1.2% in muslims; 0% in other community)

The reason for high prevalence in Hindu community is –

1) Due to high population of hindu community, this community is on greater risk of receiving infection throw blood transfusion.

2) There is taboo that on donating blood they would suffer from weakness lifelong. Therefore in state of giving blood to there love-ones they prefer to take blood from addicted professional blood donors who where mostly infected by HCV and HBV infectious diseases.

Based on the results and the discussion the following conclusions are drawn-The overall prevalence of HCV (1.6%) shows that; it may be due to low literacy rate, so the educational level of population in this region has to be increase so that they aware about these infectious disease to reduce its transmission.

1) There is need to educate the people for proper hygienic habits such that- to avoid common use of syringes, avoid intra venous drugs, proper disposable of infected biohazards, wearing gloves on handling the infected samples.

2) There is need to add of vaccination for HCV in immunization schedule to reduce its prevalence in community.

3) In hospital; medical worker has to be trained, how to exclude the professional donors so that it stop the channel of transfusion of infected blood, and also to community to motivate for blood donation.

4) In case of prostitution behavior; condoms and the other protective measures should be used to avoid blood borne diseases.

5) Younger have to be educate and discourage about harmful effects of narcotic drugs by using common syringes.

6) There is need to make severe punishable laws towards drug suppliers to protect young population.

7) There is need to organize more motivational programs to motivate blood donation, by which spread of infection can be checked, by avoiding blood donation by drug-abusers.

ACKNOWLEDGMENT:

Author were very grateful to patients and their relatives; and blood donors for their co-operation during entire study. No any funding was given by any institution or society.

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Received on 02.05.2015 Modified on 23.05.2015

Asian J. Research Chem 8(6): June 2015; Page 407-412

DOI: 10.5958/0974-4150.2015.00067.X