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A demographic study of hepatitis B in healthy population of central India.

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 Gastroenterology, 5th Edition) for centuries; epidemics of hepatitis have devastated military and civilian communities throughout the world. (2) Initially transmission of disease was attributed to nonparenteral 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 1975. (8)

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-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 Balayan (10) (In Russia, 1983) and Houghton. (11) (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 B surface antigen & hepatitis C virus infection in the blood donors attending the blood bank of Gandhi Memorial & 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 HBsAg infection among the blood donors, who are attending blood bank for blood donation; by detecting antibodies to HBsAg 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 HBsAg by rapid chromatographic immunoassay test strips (HEPAVIEW HBsAg QUALPRO DIAGNOSTICS, TULIP GROUP, GOA, INDIA). The reactive samples are further confirmed by Enzyme Linked Immunosorbent assay (Micro screen HBsAg ELISA KIT, Span Diagnostics Limited, SURAT, GUJARAT, INDIA).

Methodology of HBsAg One Step Rapid Kit Test (According to manufacturer's instruction) Rapid test kit for HBsAg is an in -vitro immunochromatographic, one step assay designed for qualitative determination of HBsAg in human serum or plasma. This test cassette contains a membrane strip, which is pre-coated with mouse monoclonal anti - Hbs capture antibody on test band region. The mouse monoclonal anti-HBs--colloid gold conjugate and serum sample moves along the membrane chromatographically to the test region (T) and forms a visible line as the antibody-antigen reaction resulting in the formation of pink/purple colour line on the test region which confirms the presence of HBsAg. The test cassette has a letter of "T" and "C" as "Test Line" and "Control Line" on the surface of the cassette.

Methodology of Microwell ELISA Method for HBsAg:

Principle: The test is based on solid phase microplate direct ELISA (Sandwich ELISA) technique. The microwells are coated with mouse monoclonal anti-HBsAg antibodies. When patient's sample is added, if HBsAg is present in the sample, it gets bound to immobilized anti-HBsAg antibodies. After removal of non-specific antigens, peroxidase labeled anti-HBsAg antibodies are added, which bind with immobilized antibody-antigen complex. After incubating and washing. TMB (Tetramethyl benzidine) substrate is added, which gives blue coloured product due to enzymatic activity. In absence of HBsAg, the colour will not change from its original pink colour. The reaction is stopped by addition of stopping reagent, which converts the blue color to end point yellow color. In a negative test, original pink colour as seen in the above step will now become colourless. The absorbance of yellow colour is directly proportional to the concentration of HBsAg present in patient's sample. It is measured by microwell plate reader having 450nm as primary filter and 630nm as reference filter.

PROCEDURE: (According to manufacturer's instruction) Bring samples and all the reagents except color reagent and conjugate to room temperature before starting the procedure:

1. Dispence 100pl of sample Diluent to required number of wells except Blank well. (leave Blank well empty)

2. Add 100 [micro]l Negative control to B, C, D, and 100 [micro]l Positive controls to E, F. Add 100 [micro]l of Samples to rest of the wells, pipette up and down for mixing, seal strips securely with Adhesive strip covers.

3. Incubate for 60 minutes at 37[degrees]C.

4. Remove and discard the Adhesive strip cover. Decant the content of wells into waste container. Fill the wells with approximately 350 [micro]l of Diluted washing Buffer and allow soak time of 30 seconds (programme the auto washer for soak time of 30 seconds) and then decant it in the waste container. Repeat the washing steps for 4 more times. Drain wells on a disposable absorbent pads or towels and tap firmly to remove excess of water.

5. Add 50 pl of Conjugate Stabiliser followed by addition of 100 pl Conjugate to each well except A (Blank well). Mix the contents of microwells by agitating the strips gently for 5-10 seconds and seal strips securely with Adhesive strip covers.

6. Incubate for 30 minutes at 37[degrees]C.

7. Repeat step 4 for washing.

8. Add 100 [micro]l of colour reagent to the all the wells including Blank well. Seal strips securely with adhesive strip covers.

9. Incubate for 30 minutes at room temperature in dark.

10. Remove and discard Adhesive strip cover and add 100 pl of stopping solution to each well. Mix the microwells by agitating the strips gently for 5-10 seconds. Blue color of Positive control/ reactive samples will turn to yellow on addition of stopping solution.

11. Read the absorbance within 30 minutes with microwell plate reader using 450nm as primary filter and 630nm as reference filter after blanking with A well(reagent well) and calculate the results.

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.

Table 1 : Prevalence of HBsAg according to type of donor

  Type of Donor      No.        HBsAg        Prevalence
                            Positive Cases       %

 Voluntary Donor    4431         137            3.0
Replacement Donor    850          14            1.6

      Total         5281         151            2.8

Statistical Inference for HBsAg: Chi-square test between voluntary and replacement donor [chi square] = 6.1 and p = 0.013 (Difference is significant since <0.05) Table 1 shows the prevalence rate of HBsAg was 2.8%. The voluntary donors accounting 3.0 % prevalence which is significantly (p< 0.05) more than replacement donors having 1.6% of prevalence.

Table-2 shows overall prevalence rate of HBsAg infection in male and female was 2.8%. The prevalence rate of HBsAg in Male (3%) was higher than Female (1.2%).

Table 3 shows prevalence of various age groups in donors. The prevalence of HBsAg in age group range between 18-28 years was 21.1%, 29-38 years was 28.4%, 39-48 years was 32.4% and 49-58 years was 17.8%. The prevalence of HBsAg was higher in age group 39-48 years it was 32.4%.

Table 4 shows the prevalence of HBsAg in donors based on their occupation. The prevalence of HBsAg in working population was 3.96%, in student it was 2.9%, in farmer it was 0.87%, and in others (laborer, house- wives etc.) it was 2.24%. Higher prevalence was found in working population.

In our study it was also found that the prevalence of HBsAg in Hindus was 2.9 % and in Muslim it was 1.6% 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 3rd generation. The overall prevalence of HBsAg infection was 2.8%.

In India, various studies have been done to determine prevalence of HBsAg with other blood bore diseases. On blood bank healthy blood donors studies; the overall prevalence of HBsAg ranges between 0.063 to 3.51%. 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.

The results of present study for HBsAg sero-prevalence was similar to findings made by Nilima sawke et al. (20) (2006-08) from Bhopal; She studied 5008 blood donors (Voluntary & Replacement) from January 2006 to December 2008 at blood bank People's College Of Medical sciences & Research Centre Bhopal by using ELISA kit and found a prevalence of 2.9% (149/5008).

The HBsAg prevalence of 2.8% seen in present study is with in the range 0.063 - 3.51 according to Indian studies. Out of the various factors which need to be considered to explain this prevalence rate with other Indian studies are the epidemiology, demographic profile and social milieu of the region. No previous studies from this region are available for comparison. The nearest studies available from central India are by Nilima sawke ET al (20) (2006-08) from Bhopal and from Dharmesh Chandra ET al. (21) (2009-13) from Gwalior, which have reported prevalence rates 2.9% and 3.51% respectively.

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 Jaiswal et al (13) 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 1.7% 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 39-48 years (32.4%) for HBsAg prevalence.

Similar findings was found by Sayed A. Qadri et al (17) in 2013 at Bijapur karnatka (31.1%) but it was more in elder group that is 51-60 years. Fathi Abed A l-Gani (23) in 2011 in Jordan found prevalence of HBsAg 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.

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

3. No vaccination by government to prevent it (Not included in national vaccination programs.)

4. 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 working population (Employed population) in case of HBsAg (3.96%).

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 groups.

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

4. There are no strict punishments 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 2.9% (And 1.69% in muslims; 0% in other community)

The resion for high prevalence in Hindu community is:

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

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

Based on the results and the discussion the following conclusions are drawn - The overall prevalence of HBsAg (2.8%) 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 HBV 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 behaviour; 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 needed 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.

DOI: 10.14260/jemds/2015/203

ACKNOWLEDGMENT: I am very grateful to my 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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(9.) Rizzetto M, Canese MG, Arico S: Immunofluorescence detection of a new antigen antibody system (Delta/anti -delta) associated with hepatitis B virus in liver and serum of HBsAg carriers. Gut 1977; 18:1997.

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(14.) Singh K, Bhat S, Shastry S, Trend in seroprevalence of hepatitis & viral infection among blood donors of coastal Karnataka, India. J. Infect Dev Ctries 2009; 3 (5): 376-379.

(15.) Bhawani Y, Rao P R, Sudhakar V, seroprevalence of transfusion transmissible infections among blood donors in a tertiary care hospital of Andhra Pradesh. Biology and Medicine 2(4): 45-48, 2010.

(16.) Kulkarni N, Analysis of the seroprevalence of HIV, HBsAg, HCV and Syphilitic infections detected in the pretranfusion blood: A short report, International Journal of Blood Transfusion and Immunohematology 2012; 2: 1-3.

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Gajendra Pal Singh Uike [1], Amar Bahadur Singh [2], Uday Raj Singh [3], S. K. Mishra [4]


[1.] Gajendra Pal Singh Ulike

[2.] Amar Bahadur Singh

[3.] Uday Raj Singh

[4.] S. K. Mishra


[1.] 3RD Year Post Graduate Student Department of Pathology, S. S. Medical College, Rewa, M.P.

[2.] 3RD Year Post Graduate Student Department of Pathology, S. S. Medical College, Rewa, M.P.

[3.] Associate Professor, Department of Pathology, S. S. Medical College, Rewa, M.P.

[4.] Associate Professor, Department of Pathology, S. S. Medical College, Rewa, M.P.


Dr. Amar Bahadur Singh, Room No.S-1, Sarla Apartment, Tala House Campus, Rewa, M. P. -486001. E-mail:

Date of Submission: 07/01/2015.

Date of Peer Review: 09/01/2015.

Date of Acceptance: 19/01/2015.

Date of Publishing: 27/01/2015.
Table 2 : Prevalence of HBsAg Positive cases according to gender

Gender     No.        HBsAg         Prevalence
                  Positive Cases       %

 Male     4623          143           3.0
Female     658           8            1.2

 Total    5281          151           2.8

Table 3 : Prevalence of HBsAg according to Age Range

Age Groups       HBsAg         Prevalence %   Male   Female
             Positive Cases

  18-28            32             21.1%        31       1
  29-38            43             28.4%        40       3
  39-48            49             32.4%        47       2
  49-58            27             17.8%        25       2

  Total            151             100%       143       8

Table 4 : Prevalence of HBsAg according to Occupation

Occupation    No.        HBsAg        Prevalence %
                     Positive Cases

 Working     1940          77             3.96
 Student     1673          49             2.9
  Farmer      912          8              0.87
  Others      756          17             2.24

  Total      5281         151             2.8

Table 5 : Indian Studies (Hbsag Prevalence Rate in Blood Donors)

SL.        Author           Year                  Place

1     Riddhi Jaiswal et   1997-2005    GSVM Medical college Kanpur
           al (13)

2      Karandeep singh     2005-07      Kasturba Medical college,
         et al (14)                              Manipal

3     Yedlapati Bhawani    2004-09         KIMS HOSPITAL(A.P.)
         et al (15)

4         Nagarekha        2005-09      Vijayanagara Institute of
        Kulkarni (16)                   Medical Sciences, Bellary

5      Sayed A. Quadri      2010        Al Ameen Medical college,
         et al (17)                              Bijapur

6      R. Shyamala et       2012         Bhaskar medical college
           al (18)                        Yenkapally Ranga Raddy

7     Sastry jayagowri     2008-13    Charitable hospital blood bank
         Met al (19)                             in Pune

8      Nilima Sawke et     2006-08     People's College Of Medical
           al (20)                      Sciences & Research Centre

9     Dharmesh Chandra     2009-13     Gajra Raja,Medical College,
         et al (21)                              Gwalior

10    Nirali shah (22)     2006-13     Sheth V.S. General Hospital,

11      Present study      2013-14       S.S.Medical College rewa

SL.        Author         No. of cases   Prevalence rate (%)

1     Riddhi Jaiswal et     1,15,073             1.7
           al (13)

2      Karandeep singh       30,428             0.74
         et al (14)

3     Yedlapati Bhawani      8,097              1.41
         et al (15)

4         Nagarekha          19,135             0.063
        Kulkarni (16)

5      Sayed A. Quadri       4,283              1.63
         et al (17)

6      R. Shyamala et        1,980              1.06
           al (18)

7     Sastry jayagowri       13,078             1.23
         Met al (19)

8      Nilima Sawke et       5,008               2.9
           al (20)

9     Dharmesh Chandra       67,132             3.51
         et al (21)

10    Nirali shah (22)       92,778             0.887

11      Present study         5281               2.8
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Article Details
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Author:Uike, Gajendra Pal Singh; Singh, Amar Bahadur; Singh, Uday Raj; Mishra, S.K.
Publication:Journal of Evolution of Medical and Dental Sciences
Article Type:Report
Geographic Code:9INDI
Date:Jan 29, 2015
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