RAJIV GANDHI UNIVERSITY OF HEALTH SCIENCES, BANGALORE,

KARNATAKA

“SEROSURVEILLANCE OF BLOOD
DONORS”
By
Dr. ANANDKUMAR GURUPADAPPA M.B.B.S.

Dissertation submitted to the

Rajiv Gandhi University of Health Sciences, Karnataka, Bangalore

In partial fulfillment
of the requirements for the degree of

DOCTOR OF MEDICINE
IN
PATHOLOGY

Under the guidance of

Dr. H. GURUBASAVARAJ M.D.
Professor

DEPARTMENT OF PATHOLOGY
J.J.M. MEDICAL COLLEGE
DAVANGERE – 577 004.

2012

I
II
III
IV
V
 ACKNOWLEDGEMENT

I thank Almighty and my family for their constant encouragement and love.

It gives me immense pleasure to express my deep sense of gratitude to

Dr. H.R. CHANDRASEKHAR M.D ., Principal, for being a constant source of

inspiration during my post- graduate training period.

My esteemed guide, Dr. H. GURUBASAVARAJ , Professor has been

M.D.

greatly instrumental in shaping this study in the present form by his advice and

meticulous approach. I am deeply indebted and greatful to him for the efforts he made

for me.

It is indeed a pleasant duty to express my feelings for Dr. S.S. HIREMATH

, Professor and Head of the Department of Pathology, JJM Medical College,

M.D.

Davangere. His knowledge and personal qualities have been highly inspirational for

me not only for this study but for whole of my postgraduation, and shall continue to

be so in the future.

I express my heartfelt thanks to Dr. PRAKASH KUMAR, M.D., Professor of

Pathology, for his constant support, guidance and blessing.

I consider myself very fortunate to have Dr. P.K. BASAVARAJA M.D .,

Dr. B.BASAVARAJU, M.D., Dr. KADAM SATYANARAYANA RAO M.D .,

Dr. RAJASHEKAR K.S., M.D., Dr. M.M. DODDIKOPPAD M.D ., Dr. K.R.

CHATURA M.D., Dr. SURESH HANAGAVADI ., Dr. ARUNA S.B.

M.D M.D ., Dr.

K.K. SURESH ., Dr. THIPPESWAMYM.T.R.

M.D ., Dr. JAYASHREE G.
M.D

PAWAR, M.D., Dr. JAGADISH C.N., ., Dr. JAGADESHWARI K.

D.C.P ., and
D.C.P

Dr. SUNILKUMAR K.B. M.D., Dr. SATISH KUMAR BELAGATTI M.D .,

Dr. SEEMA BIJJARGI M.D. , Dr. VARADENDRA KULKARANI ,

M.D.

Dr. PREETHI M.D. as my teachers who constantly helped and encouraged me during

my study period.

VI
 I am extremely thankful to Dr. JAGADESHWARI K, Blood bank officer,

Bapuji Blood bank and Mr. PRAKASH CHADICHAL, ELISA technician.

I would like thank all the technicians of Department of Pathology and Central

Lab and Blood bank for their skilled assistance and cooperation.

I am grateful to Mr. D.K. SANGAM, Biostatistician for providing help in

doing statistical analysis.

My special thanks to Dr. RASHMI REDDY, Dr. FOUZIA, Dr.

POORNIMA, Dr. CHETANA, Dr. NIDHI and Dr. SAIKUMAR, and all my post

graduate colleagues for their help and cooperation in completing this study.

I thank Mr. MAHESH, Chief Librarian and all the Staff Members of Library,

J.J.M. Medical College, for their cooperation and assistance during my study period.

I also thank to ZEN COMPUTER TECHNOLOGY for their effort in typing

and completion of this work.

I owe galaxy of thanks to my wife, PRATIBHA and my daughter

ANUSHKA and all my family members for being the constant source of

encouragement and support during my postgraduate course.

I thank all my CASES who formed the backbone of the study without whom

this study would not have been possible.

Lastly I am ever grateful to the ALMIGHTY GOD for always showering his

blessings on all of us.

VII
 LIST OF ABBREVIATIONS USED

Ab – Antibody

Ag – Antigen

AIDS – Acquired immunodeficiency

syndrome ALT – Alanine transferase

CCR5 – Chemokine receptor 5

CDC – Centre for disease

control

DNA – Deoxyribonucleic acid

ELISA – Enzyme linked immunosorbent

assay gp – Glycoprotein

HBcAg – Hepatitis B core antigen

HBsAg – Hepatitis B surface

antigen HBV – Hepatitis B virus

HCV – Hepatitis C virus

HIV – Human immunodeficiency virus

HLA – Human leukocyte antigen

ICAM1 – Intercellular adhesion molecule

1 IL – Interleukin

LAV – Lymphadenopathy associated

virus LTNPs – Long term non progressors

MHC – Major histocompatibility

MP – Malarial parasite

NACO – National AIDS control

organization NANBH – Non A Non B hepatitis

NAT – Nucleic acid test

VIII
NS – Nonstructural

PCR – Polymerase chain reaction

pfEMP1 – Plasmodium falciparum erythrocyte membrane proteins

1 PfHRP – Plasmodium falciparum histidine rich protein

PLDH – Parasite lactate dehydrogenase

PLHA – People living with HIV /

AIDS QBC – Quantified buffy coat.

RNA – Ribonucleic acid

RP – Replacement donor

RPR – Rapid plasma reagin

STD – Sexually transmitted disease

TNF – Tumor necrosis factor

TPI – Treponema pallidum

immobilization TTI – Transfusion transmissible

infections

UNAIDS – United nations joint conforence on HIV /

AIDS VCAM1 – Vascular endothelial cell adhesion molecule

1 VD – Voluntary donor

VDRL – Veneral disease research

laboratory WB– Western blot

WHO – World health organization

IX
 ABSTRACT

Background :

The evaluation of the data of the prevalence of transfusion transmissible

infections (TTI) among blood donors permits an assessment of the acqisition of the

infections in the blood donor population and consequently safety of the collected

donations. It also gives an idea for the epidemiology of these infections in the

community.

Objectives:

To study seroprevalence of HIV, HBV, HCV, Syphilis and prevalence of

malaria in blood donors.

Methods :

Total of 16,872 units of blood were collected from voluntary and replacement

donors during the study period from July 2009 to June 2011. All the blood samples

were screened for HIV, HbsAg, HCV, Syphilis and malarial parasites.

Results:

Out of the total 16,872 blood donors, replacement donors were (91.6%) more

compared to voluntary donors (8.4%). The seroprevalence of TTI was 1.85% in total

donors. The seroprevalence of HIV was 0.17% in total donors. No voluntary donor

was found to be positive for HIV. The seroprevalence for HbsAg was 1.58% in total

donors. The prevalence for HbsAg was more in replacement donors (1.68%) as

compared to voluntary donors (0.49%). The seroprevalence of HCV and syphilis was

0.09% and 0.01% respectively. No voluntary donors were positive for HCV and

Syphilis. No donors were found positive for malaria.

X
Conclusion:

The prevalence of transfusion transmissible infections was more in

replacement donors compared to voluntary donors. Hence, more emphasis should be

given to motivation of voluntary donors.

Key Words : Seroprevalence; Transfusion transmissible infections; Voluntary

donors; Replacement donors.

XI
 TABLE OF CONTENTS

Page No.

1. Introduction 1

2. Objectives 2

3. Review of literature 3-58

4. Methodology 59-75

5. Results 76-90

6. Discussion 91-102

7. Conclusion 103

8. Summary 104

9. Bibliography 105-113

10.Annexures

Proforma 114-115
Master chart 116-120

XII
 LIST OF TABLES

Sl. No Tables Page No.

1 Showing Type of blood donors 76
2 Sex wise distribution of blood donors 77
3 Age distribution of blood donors 77
4 Seroprevalence of TTI in total donors 78
5 Seroprvalence in voluntary and replacement donors 78
6 Seroprevalence of TTI 79
7 Seroprevalence of HIV in total donors 80
8 Seroprevalence of HIV in different donor category 80
9 Age -wise distribution of HIV seropositive donors 81
10 Sex wise distribution of HIV seropositive donors 81
11 Marital status in HIV positive donors 82
12 Geographic distribution of HIV positive donors 82
13 Seroprevalence of HBsAg in total donors 83
14 Seroprevalence of HBsAg in different donor categories 83
15 Age wise distribution of HBsAg positive donors 84
16 Sex wise distribution of HBsAg positive donors 84
17 Marital status in HBsAg positive donors 85
18 Geographical distribution of HBsAg positive cases 85
19 Seroprevalence of HCV in total donors 86
20 Seroprevalence of HCV in different donor categories 86
21 Age wise distribution of HCV positive donors 87
22 Sex wise distribution of HCV positive donors 87
23 Marital status in HCV positive donors 88
24 Geographical distribution of HCV positive donors 88
25 Seroprevalence of syphilis 89
26 Seroprevalence of Syphilis in different donor categories 89

XIII
 LIST OF GRAPHS

Sl. No. GRAPHS Page No.

1. Pie chart showing type of donors 76

2 Pie chart showing sex distribution of donors 77

3 Seropositivity in different types of donors / total donors 78

4 Seroprevalence of TTI 79

5 HIV Seropositivity in different types of donors / total donors 80

6 HBsAg Seropositivity in different types of donors / total

83
donors

7 HCV Seropositivity in different types of donors / total donors 86

8 Syphilis Seropositivity in different types of donors / total

89
donors

XIV
 LIST OF FIGURES

Sl. No. Figures Page No.

1 Structure of HIV Virion 9

2 The HIV genome 9

3 Serological markers of HBV 31

4 Diagrammatic representation of the hepatitis C viral (HCV) 35

genomic structure

5 Scheme of laboratory features during acute hepatitis progressing 39

to chronicity

6 ELISA Reader 74

7 ELISA instruments 74

8 ELISA Kits 75

XV
Introduction
 INTRODUCTION

Blood transfusion involves transfer of biological material from man to man.

Many infectious diseases are likely to be transmitted by blood transfusion.1

Preventing transmission of these infectious diseases through blood transfusion

presents one of the greatest challenges of transfusion medicine.1

According to NACO guidelines, all mandatory tests should be carried out on

donors blood samples for human immuno deficiency virus (HIV), hepatitis B virus

(HBV), hepatitis C virus (HCV), syphilis and malaria. The whole blood or

components from any unit that tests positive should be discarded.2

The evaluation of the data of the prevalence of the transfusion transmitted

infections (TTIS) human immunodeficiency virus (HIV), Hepatitis B virus (HBV),

hepatitis C virus (HCV), syphilis and malaria, among blood donors permits an

assessment of the acquisition of the infections in the blood donor population and

consequently the safety of the collected donations. It also gives an idea for the

epidemiology of these infections in the community.3

Voluntary non-remunerated blood donation is the source of the safest blood

supply to the transfusion service. In the Indian setup where voluntary donations are

fewer and poorly structured, safety of blood could still be compromised.4

Infectious agents that pose a serious threat to transfusion recipients are those

that persist in the circulation of asymptomatic individuals who are healthy enough to

be blood donors.1

Hence this study is undertaken to find out the seroprevalence of transfusion

transmissible infections among voluntary and replacement blood donors.

1
Objectives
 OBJECTIVES

To study seroprevalence of HIV, HBV, HCV, syphilis and prevalence of

malaria in blood donors.

Review of Literature
 REVIEW OF LITERATURE

HISTORICAL ASPECTS5 :

 1628 – English Physician William Harvey described the functions of the heart and

the circulation of blood.

 1665 – First blood transfusion of record took place by Rechard Lower. Started

with dog to dog experiments and proceed to animal to man.

 1818 – Dr. James Blundell, a British Obstetrician performed first successful man

to man transfusion for the treatment of post partum hemorrhage.

 1901 – Karl Land Steiner discovered the ABO blood groups.

 1937 – World’s first blood bank was established at Chicago ’s Cook Count y

hospital by Dr. Bernard. Fantus

 1939-India’s first Blood Bank wasset up in Kolkata.

 1940 – Karl Land Steiner and A Weiner discovered Rh blood group system.

 1968 – Screening of blood for HbsAg was introduced by Okochi et al.

 1989- India’s first AIDS patient due to blood transfusion was reported.

 2000 – World Health day 7th April 2000 was celebrated with “save blood starts

with me” as the slogan of the year.

Blood transfusion is a life saving intervention that has an essential role in

patient management within health care systems. The establishment of systems to

ensure that all donated blood is screened for transfusion – transmissible infections is a

core component of every national blood programme. Globally however there are

significant variations in the extent to which donated blood is screened. The screening

strategies adopted and the overall quality of effectiveness of the blood screening

process. As a result in many countries the recipients of blood and blood products

3
remain at unacceptable risk of acquiring life threatening infections, that could easily

be prevented.2

Transfusion medicine is relatively young field that has developed only since

the second half of the last century. These therapeutic approaches also caused the

problems of such as the incompatibility of red blood cells and plasma between donors

and recipients and possibility of transmitting infectious diseases.6

Today donor evaluation, laboratory screening tests and pathogen inactivation

procedures are considered crucial tools to reduce the risk of TTI’s but donot

completely eliminate all risk. These advances have moved transfusion medicine

towards increasingly safer products.6

The main diseases transmitted through blood are hepatitis, AIDS, syphilis and

malaria.1

In India it is mandatory to test each blood unit for HIV , HBV, HCV syphilis

and malaria.1:

HUMAN IMMUNODEFICIENCY VIRUS (HIV)

Ever since its recognition in 1981, HIV / AIDS continued to ravage all the

continents. The infection with HIV may develop to AIDS at different rates in different

individuals with a spectrum varying from rapid progression to long term non

progression. Once infected the person is affected for life. AIDS can be called as

modern pandemic affecting both industrialized and developing countries.7

4
History. 7,8,9

Historical aspects in the world

In 1981 – A new syndrome was officially recognized for the first time at

centre of disease control (CDC) USA in the previously healthy homosexual men

dying suddenly with pneumocystis carinii,now P jiroveci pneumonia and candidiasis

and also with a patient with Kaposis sarcoma.

In 1982 – the disease was named as AIDS.

In 1983 – The virus was identified by French Scientists led by Dr. Luc

Montegnier, Pasteur institute of Paris as Lymphadenopathy associated virus.

In 1984 Rober Gallo and his associates isolated the virus at national institute

of health USA and named it as Human T lympho trophic virus.

In 1985-The first blood screening test to detect HIV antibody was licensed.

In 1986 – The international committee of nomenclature of viruses has given

the common name for both LAV and HTLV-III as human immunodeficiency virus

(HIV).

In 1986-87 – A new virus of the same character has been identified in west

Africa and is now is called as HIV-2 , discovered by French worker.

In 1996- P24 antigen screening was introduced, to reduce the window period

before antibody appearance by a week

Historical aspects of India: 7,8,10

1985 – Surveillance for HIV infection was initiated in India by Indian Council Of

Medical Research (ICMR) as part of AIDS task force.

5
1986 – First report of HIV infections in sex workers in Chennai and first reports of

AIDS in Mumbai.

1989 – HIV infection reported among intravenous drug users in Manipur state.

1990-91 – National AIDS control organization (NACO) was established. Indian

national AIDS control programme was launched.

1991 – Presence of HIV-2 infection was reported for the first time from Bombay.

1992 – ICMR established national AIDS research institute (NARI) in Pune city.

1999 – The supreme court ruling made HIV testing of all blood bottles mandatory.

NACO estimate 3.87 million HIV infections in India.

2001 – Prevention of HIV transmission from mother to child using single dose

Nevirapine.

2005 – First AIDS vaccine trial was initiated.

EPIDEMIOLOGY :10

Global HIV / AIDS epidemic summary :

Global percentage of people living with HIV / AIDS (PLHA) has leveled off

since 2000.

The rate of new HIV infection was fallen in several countries but globally

these favourable trends are at least partially offset by an increase in new infections in

other countries.

Table 1 : HIV / AIDS epidemic update December 2009

Total 33.4 million (31.1 to 35.8 )

No. of PLHA Women 15.7 million (14.2 – 17.2 )
children <15 years 2.1 million (1.2 – 2.9 )
People newly infected 2.7 million (2.4 – 3.0 )
AIDS related deaths 2 million (1.7 – 2.4 )

6
 Sub Saharan Africa remains the region most heavily affected by HIV,

accounting for 22.4 million of PLHA and 1.4 million deaths in 2008.

Women account for half of the PLHA a world wide.

Young people aged 15-24 years account for an estimated 45% of new HIV

infection worldwide.

HIV / AIDS situation in India.9,11

 2.31 million (2 to 3.1) PLHA in India in 2008.

 National adult HIV prevalence was 0.36% with male HIV prevalence of 0.4%

and female HIV prevalence of 0.27%.

 HIV prevalence among general population and high risk groups varies in

different states. The adult prevalence rate of HIV infection has stabilized over

the last there years.

 84.6% of infections were transmitted through the sexual route and perinatal

transmission accounted for 4.34% of infection. 1.8% and 1.9% of infections

are acquired through injection of drug and contaminated blood and blood

products respectively.

 As per the reports of NACO (2009) the seropositivity rate in adult population

is 0.34% and in blood donors 0.28%.

Karnataka 11

 Adult HIV prevalence is less than 1% (0.75%)

 Karnataka with three other southern state contribute 63% of the PLHA of the

country.

 11% of PLHA of the country stay here.

7
Etiology : The properties of HIV

AIDS is caused by a HIV, a non transforming human retrovirus belonging to

the lentivirus family. Two genetically different but related form of HIV called HIV1

and HIV 2 have been isolated from patients with AIDS.

HIV 1 is the most common type associated with AIDS prevalent all over the

world while HIV-2 largely confined to west Africa. Although HIV-2 appears to cause

the same disease as HIV-1, it is reported to be less pathogenic.

Biology of HIV 13,14,15,16

The virion : the HIV 1 virion particle forms a ico sahedral sphere with 72

projections consisting of the envelope glycoproteins (gp) 120 and 41. Only gp41

traverses the lipid bilayer (transmembranous) the gp 120 is loosely and non covalently

bound to gp41. Under the lipid layer, the matrix protein (p17) covers the internal

surface of the viral coat. The capsid protein (p24) constitutes the internal core shell,

whereas p6 and p7 form part of nucleoid structure. The p7 protein and reverse

transcriptase (RT) molecule (p 66/51) are associated with two copies of the single

stranded genomic HIV RNA.

8
Fig. 1 : Structure of HIV Virion

Fig. 2 : The HIV genome

9
The genome :

HIV has genes that encodes the structural and regulatory proteins.

Structural genes :

gag – Encodes proteins that form core of virions p17 matrix protein, P24

capsid protein p15- nucleocapsid precursor processed to p2, p7, p1 and p6.

pol : Encodes the enzymes, RT – reverse transcriptase, PR – protease, and 1N-

integrase.

env : Encodes the envelope glycoproteins gp120 and gp41.

Regulatory :

tat : Transcriptional activator (p14) more efficient synthesis of full length transcripts.

rev : Regulator of structural gene expression (p19)

vif : Viral infectivity factor (p23) overcomes, inhibitory effects of APOBEC

preventing viral DNA degradation.

vpu : Viral proteins U, promotes, CD4 degradation efficient maturation and release of

viron.

vpr : Viral protein R’ (P15), promotes G2 cell cycle, arrest and fascilitates HIV

infection of macrophages.

nef : Negative effector (p27) down regulates cell surface CD4 and MHC 1 expression,

blocks apoptosis, enhances virion infectivity alters, the state of cellular activation

leading to productive HIV infection.

These genes are flanked by long terminal repeats which contains regulatory

elements, of gene expression.

The major difference between HIV 1 and HIV 2 ge nome is that HIV 2 lacks

the vpu gene and has vpx gene instead.

10
 Other difference is that the lipid membrane is penetrated by two types of

glycoprotein molecules gp41 and gp120 in HIV-1 and appears as small projection in

the surface of virus particles. The correspond ing glycoproteins in HIV-2 are

gp110/130 and gp36 respectively. It is the structure of these two glycoproteins which

appears to be the major difference between HIV1and HIV2.

The antibodies of these two specific set of envelope proteins do not cross-react

and form the basis for various types of tests kits.

Receptors of HIV : 17

CD4 – macrophages, monocytes, T cells B cells, dendritic cells.

Fc – Monocytes, T cells, fibroblasts, GIT, placenta

Gal c– GIT, Brain

CR-2 – dendritic cells

CD receptors – CCR5, CXCR4.

Antigenic variations and subtypes : 12,18,19

HIV is a highly mutable virus with frequent genetic variations. Based on molecular

and antigenic differences 2 types of HIVhas been recognized HIV 1 and HIV2.

HIV-1 : It expresses very high degree of molecular heterogeneity. This genetic

variability is achieved by several means like simple base substitutions, insertions,

deletions, recombination and gain or loss of glycocylation sites.

Based on env gene sequences HIV-1 has 3 distinct virus groups.

Group M : Comprises of nine subtypes or clades designated as ABCDEFGHIJK as

well as many circulating recombinant forms (CRFS).

11
 Subtypes B is predominant in USA, Canada, South America, Western Europe

and Australia. Subtype C is the most common world wide and also in India.

Group N : Very rare found only in Cameroon.

Group 0 : As also known as outlier found in Gabon, Cameroon and France.

HIV-2 has 6 subtypes A-F type A is most preva lent: HIV-2 has milder and

slower progression of the disease, with longer incubation period and is poorly

transmitted vertically.12 HIV-2 is prevalent in India.18

Heterogenisity of the virus is to be considered when developing a successful

diagnostic test, successful vaccine and a therapeutic agent to that particular

geographic area.12

Modes of HIV transmission :

Efficiency of different routes of HIV transmission and their contribution to

total number of cases :

Table 2 : Route of infection and efficiency of HIV transmission :

Percentage of efficiency Percentage of total

Route
(world over) World over India

Blood transfusion 90-95% 5 3

Perinatal 20-40 10 3

Sexual intercourse 0.1-1 75 84

Injecting drug use 0.5 – 1.0 10 3

Needle stick < 0.5 0.1 -

Others - - 7

12
I) Sexual transmission : HIV is predominantly a STD world wide. Most common

mode of infection particularly in developing countries is heterosexual transmission.

II) Transmission by blood and blood products :12

 The most efficient mode of transmission (90-100%).

 Transmission depends on the dose of virus injected. Risk is higher with

transfusion than with needle sticks.

 Blood and blood products which can transmit HIV virus are : whole blood,

packed red cells, leucocytes, platelets, fresh frozen plasma, concentrates of

clotting factors and transplanted tissues.

III) Occupational exposure 12

There is small but definite occupational risk of HIV transmission in health

workers. The risk of transmission of HIV infection following skin puncture from a

needle or sharp object is 0.3% and for mucous membrane is 0.11%. The risk of

transmission from infected health workers to patients is extremely low.

IV) Maternal – fetal / infant – transmission.18

HIV infection can be transmitted from an infected mother to her fetus in

utero,. during delivery and breast feeding. Transmission occur most commonly in

perinatal period.

REPLICATION:12,19

It includes the following steps :

1) Viral entry – adsorption; penetration and uncoating. The first step is binding of

env gp 120 with CD4 molecule of target cell. This includes conformational

change in viral envelope. Thus exposing gp41 which initiates fusion with host

13
 cell membrane. Following fusion HIV genome is uncoated and internalized

into the target cell.

2) Biosynthesis : It consists of following steps :

a) Reverse transcription and integration : Single stranded RNA genome is

converted to RNA : DNA hybrid by the viral reverse transcriptase enzyme.

Double stranded DNA is synthesized from the RNA : DNA hybrid. This is

integrated into host cell chromosome by integrase this provirus my remain

latent or it may manifest varying levels of gene expression upto active

production of virus.

b) Translation mRNA into early protein or non structural proteins. These are

enzymes which initiate and maintain synthesis of viral components. They also

shut down host cell protein and nucleic acid synthesis.

c) Synthesis of late proteins, which are the components of daughter virion

capsids.

3) Assembly and budding :

Daughter virions are formed by the assembly of HIV proteins, enzymes, and

genomic RNA at the plasma membrane. Budding of the progeny virus occurs

through lipid rafts of the host cell where it acquire its external envelope and

release mature virion.

PATHOGENESIS AND PATHOPHYSIOLOGY :

The pathogenic mechanism of HIV disease are multifactorial, and multiphasic

and are different at different stages of diseases. The events can be better appreciated

in untreated HIV infected individuals.12

14
The course of HIV infection : 19,21

Three dominant patterns of HIV disease progression have been described these

are based on the kinetics of immunologic and virologic events with body.

1) Typical progressors : This pattern is seen in 80-90% of individuals with

median survival time of 10 years. It involves three phases : Primary infection,

clinical latency and clinically apparent disease.

2) Rapid progressors: 5-10% of individuals show this pattern with median

survival time of 3-4 years; progression to AIDS over 2-3 years after

seroconversion. Immune response is defective with very low HIV specific

antibodies and CD8 cell mediated immunity.

3) Long term non progressors : (LTNPS) : 5% of individuals do not experience

disease progression for an extended period of time. They have stable CD4

cells count over long period without any therapy. Cell mediated and humo ral

immune responses are comparatively stronger. The viral load is very low. The

pathogenecity of the infecting virus and host genetic factors may be

responsible for these LTNPS.

Natural history of HIV infection: 12,18,21

The course of the disease from time of initial infection to the development of

full blown AIDS is divided into 5 stages by WHO.

1) Primary infection / acute retroviral syndrome (seroconversion). Occurs in 50-

70%. Events occurring during primary HIV infection are critical determinants

of subsequent course of HIV disease. Virus reaches the lymphnodes and other

lymphoid tissues especially gut associated lymphoid tissues (GALT) by two

modes.

15
 i) Entering directly into the blood stream (transfusion IDUS, maternal –

fetal etc)

ii) Mucosal virus entry locally (vaginal, rectum, urethra, upper GIT)

Here dendritic cells play an important role. They can trap HIV and mediate the

efficient trans infection of CD4 + T cells. Once the virus is disseminated to lymphoid

tissue, intense replication of virus occurs in activated T cells and spills to the

circulation. This occurs during first 2 - 3 weeks after infection and is called the stage

of virus dessimination, which coincides with “FLU” like illness known as acute

retroviral syndrome.

Clinically it is characterized by fever, pharyngitis, Lymphadenopathy,

arthralgia, nausea, hepatosplenomegaly, and some neurologic features.

Lab findings : lymphopenia followed by lymphocytosis with depletion of CD4

cells, high levels of viraemia and negative HIV serology.

2) Asymptomatic infection : (CD4 cell count > 500 cells/cu mm) This coincides

with robust and intense immune response by the host. Both effective cell

mediated (HIV specific cytotoxic lymphocytes) and humural response

(complement and neutralizing antibodies) come into play.

The period from HIV entry in the host and appearance of detectable levels of

HIV specific antibody is called “Window period”. It ranges from 3 weeks to 3

months on an average.

The immune response succeeds in downregulating viraemia but HIV is never

completely eliminated. The quality of T cell response determines progression

of disease further which is generally determined. There is ongoing viral

16
 replication and destruction. Because of this rapid viral proliferation it is

estimated that every possible HIV genome mutates on daily basis.

3) Symptomatic HIV infection : (CD4 count 200-499 / cu mm)

There is gradual depletion of cell mediated immune response and increasing

levels of viraemia shows early symptoms of HIV related illness like oral

thrush, diarrhea, weight loss, low grade fever, herpes zoster etc.

4) Late stage HIV disease (CD4 count 50-200 / cu mm)

Signs and symptoms of AIDS defining illness are seen. It is characterized by

opportunistic infections and malignancies, like pneumocystis pneumonia,

toxoplasmosis, tuberculosis cryptococcal meningitis, kaposi’s sarcoma and B

cell lymphoma.

5) Advanced HIV disease (CD4 count < 50/ cumm)

Continuation and exacerbation of AIDS defining illness with CNS

involvement like AIDS dementia complex and CNS lymphomas. AIDS

wasting syndrome with weight loss > 10% in less than 4 weeks.

Serological profile of HIV infection:22

Window period : Defined as the period from entry of HIV in the host and appearance

of detectable level of HIV specific antibodies. It is usually 4-6 weeks in case of

transfusion of infected blood during which infection establishes. During the period

individual is infectious but has no demonstrable antibody. Viral antigen (p24) may be

demonstrated in some. The window period may be prolonged after infection from

sexual route.

IgM antibodies appear in 4-6 weeks, followed by IgG antibodies to different

parts of virus (antigen) including core proteins and envelope glycoprotein. Soon after

17
the appearance of antibodies there is decrease in the level of free antigen, but the

production of p24 antigen never ceases completely. Normally the level of antibodies

to p24 and envelope glycoprotein gp-41 peaks and persists through out the

asymptomatic phase of HIV disease. However antibodies to p24 may disappear and

p24 antigen may becomes detectable again as HIV disease progresses into AIDS.

Laboratory diagnosis of HIV infection :

Detection of antibodies to HIV-1 and HIV-2 is an accepted method for

identification of HIV infected blood donations.22

The diagnosis of HIV infection depends upon the demonstration of antibody to

HIV and the direct detection of HIV antigen or its components. The laboratory aspects

of HIV diagnosis is two step process which requires sequential use of highly sensitive

screening test followed by a highly specific confirmatory assay : 14

Test to detect specific antibody can be classified into :

1) Screening tests.21

a) ELISA

b) Simple / Rapid tests

 Particle agglutination

 Membrane enzyme immune assay

 Immunochromatography assay.

2) Supplemental or confirmatory tests :15

Used for diagnosis of HIV :

 ELISA and rapid tests with different antigen system

 Western blot (WB)

 Line immunoassay (LIA)

18
  Indirect fluoroscent antibody test (IFA)

 Radio immuno assay (RIA)

3) Other tests21

 P24 antigen assay

 Cultures

 Polymerase chain reaction (PCR)

 Plasma viral load by nucleic acid test (NAT)

ELISA :13,21

It is the most commonly performed screening assay in blood banks and tertiary

care hospitals. Easy to perform adaptable to large number of samples. Sensitive,

specific and cost effective.

Different generations of ELISA kits :

First Generation : Use antigen derived from viral lysates

Second Generation : Use artificially derived recombinant antigens expressed from

bacteria or fungi; Window period is 42 days.

Third Generation : Use chemically synthesized oligopeptides and employes a

sandwich technology using enzyme coupled HIV antigen with improved specificity.

Window period is narrowed down to 22 days.

Fourth generation : Combination of standard third generation ELISA with p24

antigen detection assay. Improves HIV detection during HIV seroconversion.

Reduces window period to 16 days.

19
Principles of ELISA :21

 Indirect ELISA

 Competitive ELISA

 Sandwich ELISA

 Capture assay.

All ELISA consists of either HIV Ag or Ab, solid phase (matrix or support)

conjugate and substrate detection system. The color produced is measured on a

ELISA reader as optical density values (OD). Kit controls and internal controls must

be included with each round to ensure quality results.

Indirect ELISA:21

HIV antigens are attached to the solid phase allowing antibodies to bind and

are subsequently detected by enzyme labelled anti human immunoglobulin and

specific substrate system. It is the most commonly used.

Competitive ELISA :21 Here antibodies in the specimen compete with the enzyme

conjugated antibodies in the reagent to bind with antigen on solid phase. Hence faint

or no color is produced if antibodies are present. Development of strong color means

it is non reactive.15

Antigen and antibody sandwich ELISA :21

Modification of indirect ELISA to improve sensitivity and specificity as on

solid phase binds to antibody of test specimen.These multivalent Ab molecules will

again bind to enzyme labelled Ag in reagent. Thus a sandwich of Ag+ Ab + Ag

(enzyme labeled) is formed. This can be detected by adding suitable substrates.15

20
Antigen / Antibody capture assay :21

This is based on either indirect or competitive ELISA : A monoclonal Ab is

attached to solid phase. HIV Ag supplied as reagent is added which binds to

monoclonal antibody. Now test serum is added if antibody is present, it will attach to

HIV Ag. It is more specific than direct ELISA.15

False positive ELISA results :12,13

Seen in autoimmune diseases, multiple transfusions, hypergamma

globulinemia, chronic alcoholics, multiple pregnancies, antibodies to class II HLA –

Ag, technical errors, cross reactive antibodies with hepatitis B virus, malaria and

dengue.

False Negative ELISA :12,13 Seen in window period, immuno suppressive therapy,

late stage disease, (immune collapse), B cell dysfunction, and technical errors.

Rapid assays :12

They are based on principles of agglutination. ELISA, or immuno

chromatography. Generally requires less than 30 minutes and do not require special

equipments or trained personnel.

Agglutination assay :14 Incorporates variety of Ag coated carriers like red cells, latex

particles, gelatin and microbeads. HIV antibody if present, agglutinates the particles

which are coated with Ag and a sort of lattice network is fo rmed which can be

visualized macroscopically. They have very good sensitivity, easy to perform,no wash

procedures and are cost effective. However specificity is somewhat compromised.8

Immunochromatographic assay : This employ lateral or capillary flow of sample

along a solid support to permit interaction with an embedded Ag and is detected by

21
adding appropriate conjugate and substrates which gives a visible coloured compound

as dots or lines. Controls are included to identify non specific reactivity. They can

usually descriminate between HIV-1 and HIV-2. Sensitivity and specificity compares

with ELISA. Only draw back is the high cost.13,14

Choice of screening test depends on the21

 Objective of testing

 Sensitivity and specificity of assay

 Prevalence of HIV infection in the population.

 Cost effectiveness.

Supplemental / confirmatory assay :7,14,23

Western Blot (WB): It is considered as the gold standard for the validation of

HIV antibody reactive samples most widely accepted and highly specific. May give

indeterminate results.

This test detects the antibodies to various components of HIV. The initial step

in the Western blot test is the production of immunoblot. Detergent disrupted purified

HIV is separated into various proteins (antigens) according to their molecular weight

by electrophoresis on polyacrylamide slab gel in the presence of sodium dodecyl

sulfate (SDS). The separate HIV proteins are transferred to solid support membrane

usually nitrocellulose membrane. The antigen impregnated nitrocellulose membrane

(blotted membrane) is then cut into strips, each strip having full range of viral proteins

which were separated in the gel.

22
Interpretation of results :14

Positive result : Western blot test to be called positive, various criteria are

recommended by different organizations.

Table 5 : Criteria for positive western blot :

Organization Criteria (for positive WB test)

WHO 2 env (gp120, 160, 41) with or without gag/pol
CDC Any two p24, gp41 gp120/160.
Dupont P24, p31, and gp41 or gp120/160.

Negative test : No band on intracellulose structure.

Indeterminate test : Bands present but doesnot satisfy the criteria.

P24 antigen assay :

Although the detection of HIV antigen has limited value presently for blood

transfusio n practice. It is useful for diagnosis during window period and in newborn.

p24 is one of the earliest viral antigens detectable in the blood. Levels of free antigen

decline after the appearance of anti p24 antibodies and formation of immune

complexes. An increase in the p24 levels is a predictor of progression.

This assay uses ELISA principle. Sensitivity is enhanced by including immune

complex dissociation at acid pH, quantification for monitoring p24 and is possible by

testing serial dilution of plasma. The window period reduced to 15 days.

Nucleic acid amplification technology (NAT) test :

They usually detect the presence of viral DNA or RNA by employing various

techniques like PCR, (polymerase chain reaction), branched DNA assay and nucleic

acid sequence based amplification. The window period reduced to approximately 5.6

days

23
WHO strategies / algorithms for HIV testing :

UNAIDS / and WHO recommendations for HIV testing strategies according to test

objective and prevalence of infection in the sample population.

Table 3 : Selection of testing strategy

Objective of testing Prevalence of infection Testing strategy

applicable.
Screening of blood and All I
blood products
Surveillance > 10% I
< 10% II
Diagnosis
a) with clinical signs and >30% I
symptoms  30% II
b) asymptomatic > 10% I
<10% III

In India presently WHO strategy I is used for screening blood donors for HIV

antibodies. According to this strategy, serum / plasma of each and every donor blood

unit is tested by highly sensitive ELISA / rapid / simple assay for HIV-1 and 2

antibodies. If the test is negative the unit is considered free of HIV and if reactive the

unit is discarded.21

HEPATITIS B :

Transfusion related hepatitis is almost exclusively caused by viruses. Hepatitis

B is the most important causative agent of transfusion associated hepatitis and is

associated with protracted carrier state and chronic liver disease. Chronic carriers of

HBV are at risk of long term seque lae and may progress to cirrhosis, liver failure and

hepatocellular carcinoma.24

24
History :25,26

1943 – Transmission of hepatitis through blood transfusion was first time reported.

1965 – Blemberg et al while studying human serum proteins in the serum of an

Australian aborigine observed new antigen which gave a clearly defined line of

precipitation with sera from two hemophilia who had received multiple blood

transfusions. This was named Australian antigen.

1968 – Australian antigen was found to be associated with serum hepatitis and was

subsequently named as HBsAg.

1969 – Laboratory screening for HBsAg began with testing of blood donor.

1970 – Dane detected a double walled spherical structure, 42 nm in diameter. This is

complete HBV and is known as Dane particle.

Epidemiology :24,27

Global :

The HBV infection si a global problem, with 66 percent of all the world’s

population is living in areas where there are high levels of infection.

According to WHO estimates 2 billion people have been infected with HBV,

of whom 350 millions are chronic carriers, and 4 million new infections occurs

annually.21

Internatioal Task force on hepatitis B has classified geographical areas into,

low (< 2%) intermediate (2-7%) and high prevalence regions (>7%).28

The risk of HBV transmission by blood transfusion in USA is 1:63,000 units

transfused:24

25
 The prevalence of HBsAg positivity in donor population in USA and UK is

about 0.1%.24

Percentage seropositivity of HBV in indian study was shown to be 1.55% in

1996. which came down to 0.99% in 2002.24

Biology of HBV :29,30,31

HBV is a DNA virus of the hepadnaviridae family of DNA viruses that cause

hepatitis in multiple animal species. There are about 8 genotypes with geographic

distribution around the globe.

The mature HBV virion is a 42 nm spherical double layered. “Dane particle”

that has an outer surface envelope of proteins, lipid and carbohydrate enclosing an

electron-dense, 28 nm slightly hexagonal core.

The genome of HBV is a partially double stranded circular DNA molecule

having 3200 nucleotides. The HBV genome contains four open reading frames coding

for.

 Nucleocapsid core protein (HBc Ag, hepatitis B core antigen) and a longer

polyp eptide. Transcript with a precore and a core region designated HBeAg

(hepatitis ‘B’ “e” antigen). The precore region directs the HBeAg polypeptide

towards secretion into blood, where as HBcAg remains in hepatocytes for the

assembly of complete virions.

 Envelope glycoprotiens (HBsAg) which consists of three related proteins;

large HBsAg (containing pre S1, pre S2 and S) middle HBsAg (containing pre

S2 and S) and small HBsAg (containing S only). Infected hepatocytes are

26
 capable of synthesizing and secreting massive quantities of non infective

surface protein (mainly small HBsAg)

 The polymerase (Pol) that exhibits both DNA polymerase activity, genomic

replication occurs via an intermediate RNA template through a unique

replication cycle DNA  RNA  DNA.

 HBX protein which is necessary for virus replication and may act as

transcriptional transactivator of the viral genes and wide variety of host genes.

It has been implicated in the pathogenesis of liver cancer in HBV infection.

Genotypes :

Molecular subtyping of HBV :

HBsAg exhibits antigenic diversity. It contains two different antigenic

components the common group reactive antigen, ‘a’, two pairs of type specific

antigens d-y and w-r. Only one member of each pair is present at a time. HBV thus

can be subdivided into 4 major antigenic subtypes, which are of epidemiological

importance adw, adr, ayw, ayr.

Molecular variants :29,31

Two types of mutants are commonly recognized.

1) Precore mutant : They have mutated strains of HBV that donot produce

HBeAg but are replication competent and express HBc Ag. The HBe Ag may

be low or undetected despite the presence of HBV viral load. This is

associated with fulminant hepatitis.

2) Escape mutants : Vaccine induced escape mutants which replicate in the

presence of vaccine induced immunity.

27
Replication :31

HBV attaches to the surface of hepatocytes via pre-S region of HBsAg. After

penetration and uncoating host enzymes convert partial dsDNA to covalent closed

circular DNA in the nucleus. This is converted to mRNA and pregenome RNA. From

mRNA structural and non structural proteins are synthesized. Pregenome RNA is

converted to DNA by reverse transcriptase enzyme. Encapsidation occurs and is

released through budding. These may go and infect other hepatocytes.

Modes of transmission :30,31,39

1. Parenteral route : May result from transfusion of blood and blood products :

through contaminated needles syringes and knifes, tattoing, intravenous drug

abuse.

2. Perinatal transmission : It usually occurs during birth or in the immediate post

natal period : It accounts for 90% cases in high prevalence regions.

3. Sexual transmission : Common mode of transmission in low prevalence regions

due to unprotected heterosexual or monosexual inter course : Male homosexuals

are at higher risk of acquiring infection mostly in developed countries.

4. Direct contact : By open skin lesions or with cuts or grazes especially among

young children in developing countries.

Pathogenesis:31

HBV is not directly cytopathic to hepatocytes. It is immune mediated.

Cytotoxic T cells recognize the viral antigens on the hepatocytes and destroy those

hepatocytes. Thus in the absence of adequate immune response, HBV may not cause

hepatitis but may lead to carrier state. Hence infants and immunodeficient persons are

more likely to become asymptomatic carriers.

28
Clinical presentation :29,33

Incubation period : HBV has prolonged incubation period usually 4-26 weeks.

HBV can produce :

1) Acute hepatitis with recovery and clearance

2) Non progressive chronic hepatitis

3) Progressive chronic disease ending in cirrhosis

4) Fulminant hepatitis with massive liver necrosis

5) An asymptomatic carrier state.

HBV induced chronic liver disease is an important precursor for development of

hepatocellular carcinoma.

The carrier state:28,30

Two types of carrier states are seen :

1) Simple carrier (healthy carrier) : They are of low infectivity with low titre

HBsAg and negative for HBeg, HBV and DNA polymerase

2) Super carriers : Highly infectious with high titre of HBsAg, DNA polymerase

and HBeAg in circulation.

In non-endemic areas such as united states less than 1% of HBV infections acquired

by adults produce carrier state.

In endemic areas the carrier state is seen in > 90% of cases.

SEROLOGICAL MARKERS OF HBV:28,31,32,39

Natural course of the disease can be followed by serum markers

1) HBsAg: First marker to appear in blood, being detectable even before the

onset of clinical illness. It remains throughout symptomatic course of disease

29
 with peak during preicteric phase. It may disappear with recovery of illness.

However it may persists for years in carriers.

2) Anti HbS : Does not appear until the acute disease is over and usually not

detectable for few weeks to several months after the disappearance of HBsAg :

Anti HBs may persists for life confering protection this is the basis for current

vaccination strategies using non infectious HBsAg.

3) HbcAg: HBcAg is not detectable in serum as it is enclosed within the

envelope. But can be demonstrated in cells by immunofluorescence.

4) Anti-HBc: IgM anti-HBc becomes detectable in serum shortly before the onset

of symptoms, concurrent with the onset of elevated serum aminotransfarase

levels. (indication of hepatocyte destruction). Over a period of month IgM

anti-HBC antibody is replaced by IgG anti- HBc. There is no direct assay for

IgG anti-HBc, but its presence is inferred from decline of IgM anti HBC in the

face of rising levels of total anti HBc.

5) HBeAg : HBeAg appears concurrently with HBsAg but disappears in few

weeks. Circulating HbeAg is a indicator of high infectivity.

6) Anti HBeAg : Indicates low infectivity appears after HBeAg disappears.

7) HBV DNA and DNA polymerase : Appears soon after HBsAg. Detected by

molecular methods like, polymerase chain reaction and hybridization

technique. It is indicator of infectivity and viral replication in liver.

30
 Fig. 3 : Serological markers of HBV

Biochemical markers :

 Serum bilirubin : raised

 Increased transaminase level

Occur in some case close to the peak levels of HBsAg levels in the serum.

Screening tests for HBV :28,36,39

In order to reduce the risk of post transfusion hepatitis B infection, HBsAg

screening was first of all attempted by Okochi et al at the university of Tokyo hospital

in 1968: Donated blood was screened by immunodiffusion technique for the presence

of HBsAg.

Various screening methods for detection of HBsAge and their relative

sensitivity.

31
Table 4 : Methods for the detection of HBsAg in order of sensitivity :
Tests Sensitivity
(minimum detectable level of
HBsAg)
 Immunodiffusion
 Immunoosmoelectrophorosis Low(100g/ml)

 Latex agglutination (LA)

 Reverse passive hemagglutination Moderate (10-
 Chromatographic immunoassay 100 ng/ml)
 Enzyme immunoassay High (0.030 – 0.200)
(EIA/ELISA)
(0.5 – 10 ng/ml)
 Radioimmunoassay

 Currently third generation ELISA is most widely used test for detection of

HBsAg. It is highly sensitive and specific test and detects HBsAg at as low as 0.5

ng/dl. But still the risk of post transfusion is not eliminated because the minimum

level of detection of HBsAg by 3rd generation assay system is more than 100 times

the minimum infective dose of HBV.

Nucleic acid amplification (NAT) test :

Minipool testing for HBA DNA is particularly helpful in window period. It

has reduced the window period to 24 days.

Table 5: Commonly encountered serologic patterns of hepatitis B infection

32
HEPATITIS C

Hepatitis C :

Hepatitis C was the most common, infection complication, of blood

transfusion in united states for many years. Studies in 1970 showed that more than

10% of blood recepients had biochemical evidence of what has then termed Non A

Non B hepatitis (NANBH). Subsequently all these cases were shown to be due to

infection with hepatitis C virus.

Implementation of increasingly sensitive tests for HCV infection has now

reduced the incidence of hepatitis C infection to levels that are essentially un

detectable by direct study.41

History : 42,43

In 1970 – In an effort to reduce the number of transfusion transmitted NANB viral

hepatitis infection surrogate or substantiate testing of donor blood was evaluated in

USA.

In 1974-75- The unidentified non A – non B hepatitis virus was believed to be the

cause of large number of transfusion transmitted infection.

In 1981 – an association between elevated serum levels of alanine amino transferase

in blood donor and transfusion transmitted NANB hepatitis in recipients was reported.

In 1984-86 – Antibody to the hepatitis B core antigen in blood donors was also found

to be associated with an increased risk of transfusion transmitted NANB in recipients.

In 1986 – Implementation of surrogate marker testing of blood donor using ALT and

anti HBC was instituted in the USA.

In 1989 – cloning of hepatitis C virus genome the major agent of parenterally

acquired Non A Non B hepatitis.

33
In 1990 – Anti HCV antibody tests were available for screening blood donors.

In 2001 – Screening for HCV infection of donated blood made mandatory in India.

Epidemiology :

HCV infection is seen only in humans. The source of infection is the large

number of carriers estimated to be about 200 millions world wide. 44The prevalence of

HCV antibodies in blood donors in developed countries ranges from 0.4 – 2%. The

prevalence of HCV in Indian blood donors has not been studied extensively, but as

per some available reports seroprevalence of HCV ranges between 0.4 and 1.09%. 24

The recent risk estimate of HCV is 1:103,000 per donor exposure in the US. 18 With

the implementation of NAT based HCV screening there has been a major decline in

the risk of HCV transmission to 1:3,68,000 units transfused in US.

A recent study in US has shown risk of HCV infection with minimpool NAT

screening to be as low as 1 in 2 million.41

Virology of HCV :28,39,45

HCV is a linear, single stranded positive sense 9600-nucleotide RNA virus.

The genome of which is similar in organization to that of flaviviruses and

pestiviruses. HCV is the only member of the genus hepacivirus in the family

flaviviridae.

The HCV genome contains a single large open reading frame (gene) that codes

for a virus polyprotein of ~ 3000 aminoacids which is cleaved after translation to

yield 10 viral proteins.

The 5’ end of the genome consists of an untranslated region (containing an

internal ribosomal entry) adjacent to the genes for four structural proteins : the

34
nucleocapsid core protein c, two envelope glycoproteins, E1 and E2 and a membrane

protein P7. The 5’ untranslated region and core gene are highly conserved among

genotypes.

But the envelope proteins are coded for by the hypervariable region which

varies from isolate to isolate and may allow the virus to evade host immunologic

containment directed at accessible virus envelope proteins.

The 3’ end of genome also include an untranslated region and contains the

gene for six (6) non structural (NS) proteins : NS2, NS3, NS4A, NS4B, NS5A, and

NS5B. The NS2 cysteine protease cleaves NS3 from NS2, and NS3-4A serine

protease cleaves all the down stream proteins from the polyprotein.

Fig. 4 : Diagrammatic representation of the hepatitis C viral (HCV)

genomic structure

Important NS proteins involved in virus replication include the NS3 helicage,

NS3-NS4A serine protease and the NS5B RNA dependent RNA polymerase. Because

HCV doesnot replicate via a DNA intermediate it doesnot integrate into the host

genome. Because HCV tends to circulate in relatively low titre, 103-107 virions / mL.

visualization of virus particles estimated to be 40-60 nm in diameter remains difficult,

still the replication rate of HCV is very high 1012 virions / day. Its half life is 2.7 hrs.

Complete replication of HCV and intact 55 nm virions has been described in cell

culture systems. HCV gains entry into the hepatocyte viaCD81 receptors.

35
Genotypes :38

At least six distinct genotypes as well as > 50 subtypes within genotypes of

HCV ha ve been identified by nucleotide sequencing. HCV circulates as a population

of divergent but closely related variants known as quacispecies.

Type: 1, 2, 3 predominates in North Europe and North America.

Type 4 : Is the principle genotype in middle east central and North Africa.

Type 5 : In south Africa different genotypes may influence both progression of HCV

and response to treatment. Type 1 is the fastest progressing genotype and the least

responsive to treatment.

The genotypic and quasispecies diversity of HCV resulting from its high

mutation rate interferes with effective humoral immunity.

Neutralizing antibodies to HCV have been demonstrated but they tend to be

short lived and HCV infection doesnot induce lasting immunity against reinfection

with different virus isolates or even the same virus isolate.

The E2 protein of the envelope is the target of many anti HCV antibodies but

is also the most variable region of the entire viral genome enabling emergent virus to

escape from the neutralizing antibodies. The genomic instability and antigenic

variability have seriously hampered efforts to develop HCV vaccine.

Diagnostic tests were based on antibodies to an expressed proteins (C 100-3)

derived from the NS3/NS4 region. This antigen represents 363 amino acids of viral

sequences from NS4 region.

The second series of non structural antigens c33 and c200 have been derived

from NS3 and NS4 regions. NS5 epitopes have been incorporated in third generation

36
ELISA. Antibodies to core antigen anti – c22 usually appear earlier than these to

c100-3. It is identified in blood donors with chronic NANB hepatitis who were

negative for anti c100-3. The clearance of IgM anti HCV may distinguish between

acute, resolving disease and chronic disease.

Four HCV antigens used in RIBA (recombinent immunoblot assay) comprise

one structural (c22) and three non-structural antigens (c33, c100-3, 5-1-1).

Replication :38

The viral genome of positive stranded RNA virus function as an mRNA in

infected cells. Viral proteins are initially translated in an HCV infected cell from

genomic RNA. During replication process HCV needs to synthesize a negative

stranded RNA or use as a template for generating progeny virion RNA’s. Specific

detection of HCV negative stranded RNA is important for the diagnostic purpose of

detecting replicating virus in cells.

Examination of HCV RNA by insitu hybridization in HCV infected liver

specimens shows that hepatocytes as well as mononuclear liver cells support HCV

replication. Replication of HCV can also be found in peripheral blood mononuclear

cells by detecting positive and negative stranded DNA.

MODES OF TRANSMISSION :35,38

Although the precise mode of acquisition of hepatitis C virus is often

uncertain, it is known to be transmitted by parenteral or inapparent parenteral contact

routes.

1) Parenteral : The virus circulates in relatively low titre in blood. But transmission

by blood transfusion, blood products (including factor VIII, factor IX fibrinogen and

37
cryoglobin) has been unequivocally documented. Similarly transmission among

intravenous drug abusers through shared needle account for high prevalence of HCV

infection. Needle stick injury, tattoo, ear piercing, scarifiction can also transmit. The

principle route of HCV transmission world wide is percutaneous exposure to HCV

containing blood.

Sexual transmission : The risk of transmission in multiple sexual partners and

persons with sexually transmitted diseases is 10%.35

Perinatal transmission : HCV infection occurs in 2.8% of infants born to HCV

infected mothers. This risk increases if mother is also HIV infected or if the maternal

level of HCV RNA is high. There is no conclusive evidence that HCV is transmitted

by breast feeding.Perinatal transmission most likely to play some part in maintainance

of HCV reservoir.

Others : Group with increased frequency of HCV include patients who require

hemodialysis and organ transplantation and those who require transfusion in the

setting of cancer chemotherapy.

Pathogenesis :29,38

Cell mediated immune responses and elaboration by T cells of antiviral

cytokines contribute to the containment of infection and pathogenesis of liver injury

associated with hepatitis C.

Yet consensus has emerged supporting a role in the pathogenesis of HCV –

associated liver injury of virus activated CD4 helper T cells that stimulate the

cytokines they elaborate, HCV specific CD8 cytotoxic T cells. These responses

appear to be more robust (higher in number, more diverse in viral antigen specificity,

38
more functionally effective and more long lasting) in those who recover from HCV

than in those who have chronic infection. Several HLA alleles have been linked with

self limited hepatitis C. The establishment of persistent infection correlates with

failure of adaptive immune responses to HCV. Further more HCV proteins have been

shown to interfere with innate immunity by resulting in blocking of type 1 interferon

responses and inhibition of interferon signaling and effector molecules in the

interferon signaling cascade. Also shown to contribute to limiting HCV infection are

natural killer cells of the innate immune system which function when HLA class I

molecules required for successful adaptive immunity are under exposure.

Clinical features of HCV infection :28,38

 Long incubation period of 2-26 weeks.

 Acute illness is usually mild or anicteric

 Overt jaundice is seen in 5% of cases only

 50-80% of patient progresses to chronic hepatitis with some

progressing to cirrho sis and hepatocellular carcinoma.

Fig. 5 :Scheme of laboratory features during acute hepatitis

progressing to chronicity.

39
HIV coinfection :29

Because of similar transmission mode and the similar high risk patient

population co- infection of HIV and hepatitis viruses is becoming a common clinical

problem.

Among HIV patients 10% are infected with HBV and 30% with HCV.

Chronic HBV and HCV infection is now leading cause of morbidity and mortality for

HIV infected patients and liver diseases is the common causes of death in individuals

with AIDS. HIV infection significantly exacerbates the severity of liver disease

caused by HBV or HCV. Less clear is the impact of HBV or HCV on HIV.

Laboratory diagnosis of HCV:38,46,47

Detection of antibodies to HCV is used for serodiagnosis of HCV infection :

1) First generation ELISA : Introduced in 1990: which detect antibodies to

C100-3, and part of NS4 region had relatively low sensitivity (80%).

2) Second generation ELISA : Were introduced in 1992 which included

antigens from core and NS3 region (C22c and C200 from NS3-NS4/c33c from

NS3 alone) in addition to C100-3. The ELISA-2 test was used for blood

screening in the united states until the introduction of ELISA 3 in 1997.

3) Third generation ELISA : Currently commonly used in blood banks for

screening donors units. Incorporates proteins from core, NS3 and NS5 regions

detects anti HCV antibodies during acute infection.

4) Fourth generation ELISA : Recently launched detects both the capsid Ag

and the antibodies and there by reduce the window period considerably.

Surrogate markers : Determination of Alanine amino transferase and antibodies to

hepatitis core antigen (Anti-HBc) have been used initially to identify high risk donors

40
for transmission of NANB hepatitis (HCV). Surrogate markers played important role

in the prevention of HCV prior to introduction of HCV specific assays. But presently

the values of these tests remains inconclusive and controversial.

Supplement immunoassay : one licenced supplementary test for anti HCV is

available in USA. This test is an immuno assay consisting of nitro cellulose paper

strip bearing HCV peptides at specified locations. There are also controls to ensure

that the test is performed properly and to identify reactions due to antibodies to

superoxide desmutase (SOD) a carrier protein for expression of some of the peptides.

The strip is exposed to test sample and any adherent antibodies are detected and

visualized by use of an antiglobulin conjugate and a chromogenic substrate. The test

is designed to complement 3rd generation EIA and consequently uses essentially the

same peptides. The test was developed to dissect out and identify the presence of

antibodies to different viral epitopes.

A positive test is defined on the basis of atleast two bands with density greater

than or equal to that of the low positive control but in the absence an SOD band.

Recombinant immunoblot assay : Uses the HCV antigen affixed to solid phase and

has high specificity rate : the detection of antibodies to HCV includes : bands: 5-1-1,

C100-3 C22-C, C33C and NS5 polypeptides. If antibodies to 2 or more antigens are

present the sample is considered positive. RIBA is useful in patients with positive

ELISA in whom there is dout about the diagnosis. Eg: Patient with normal ALT.

Nucleic acid amplification testing :46

Two commercially available NAT procedures are in use for testing donor

blood for evidence of HCV infectivity.

41
 One is PCR procedure that is conducted on RNA extracted by standard

chemical method from a pool of 24 plasma samples.

The other approach uses a transcription mediated amplification (TMA)

procedure on nucleid acid preparations made by a solid phase, probe capture method.

Both methods are successful in detecting HCV antibody non reactive, RNA

positive window phase donation. Detection rate for HCV RNA is about 1 in every

2,30,000 sero negative donations. The window period with NAT test for HCV is

reduced to 5 days.

HCV core antigen :46 Immunologic test for HCV core antigen have been developed

with a sensitivity that is approximately equivalent to the detection of 30,000 to 50,000

copies of HCV RNA. Although less sensitive than NAT for HCV RNA; core antigen

tests nevertheless are capable of substantially reducing the infectious window period.

SYPHILIS :

Syphilis, a chronic systemic infection caused by Treponema pallidum.

Subspecies pallidum is usually sexually transmitted and is characterized by episodes

of active disease, interrupted by period of latency.48

Syphilis is one of the earliest transfusion hazard recognized. Blood transfusion

is a potential route of infection if fresh infected blood is transfused.43

Epidemiology :

Global world wide annually an estimated 12 million cases occur, of which 4

million cases occur in Africa.

42
 In India constant decline in syphilis has been observed in recent years. In India

prevalence of VDRL reactivity varies from 0.8 to 2.7%

In a study at Chandigarh a major decline in syphilis is observed with

prevalence decreasing from 10.4% in 1977-85 to 2% in 1995-96.49

In 1940 syphilis screening was introduced in blood bank.43

 Treponema palladum causing syphilis survive at the most 5 days in

blood stored at 40C only. Rare cases of transfusion transmitted have been

documented.24

 Being sexually transmitted disease its presence points toward “high risk”

behaviour and consequence of higher risk of exposure to infections like

HIV and hepatitis.24

The chances of transfusion of syphilis have been greatly reduced because of

following reasons.28

 Exclusion of high risk donors.

 Screening for syphilis

 Storage of blood at 2-40C for 72 hours prior to transfusion

 Most of the patients who need transfusion of blood or blood products are

on antibiotic therapy.

Morphology:50,51

Treponema pallidum is a thin delicate, pale motile regularly coiled spiral

spirochete with tapering ends. Length is 8-16 m and 0.1 to 0.15 m width.

Wavelength of organism is about 0.9 m with amplitude of 0.2 m. It has 8-20 coils

at regular intervals of 1m. The terminal part is formed by an acrone like nose piece

which is responsible for attachment of organism.

43
 The cytoplasm is enveloped by trilaminar cytoplasm membrane enclosed by

cell wall. At each end of organism there are 3-4 endoflagella present in the

periplasmic space; these are responsible for cork screw motility of the organism.

Cultivation :52,53

It cannot be grown on artificial culture media, but poor yields have been

obtained in tissue culture cells. Therefore only practical method of culture is

inoculation in rabbit testis.

The discovery that T.pallidum is microaerophilic rather than a strict anaerobe

has aided the prolonged survival of the organism in vitro.

Antigenic structure :54

Based on the production of antibodies the treponemal antigens are divided into

I) Specific antigens :

1) Group specific Ag : All pathogenic and non pathogenic treponemes posses a

common group antigen. The antibody to this can be detected by reiter

treponema Ag.

2) Species specific antigen : It is poly sacharide in nature and is species specific.

II) Non specific antigens (Reagin antibody)

The antibody to this appears in blood which reacts with hapten (cardiolipin)

extracted from infected beef heart.

Modes of transmission : 51,55

1) Sexual transmission : Most common mode of transmission. Nearly 30% of the

sex partners of persons in the primary and secondary syphilis develop the

disease.

44
 2) Parenteral transmission : By blood and blood products, and accidental

inoculation while working in lab.

3) Transplacental transmission : A woman with early syphilis can infect her fetus

much more commonly. Breast feeding doesnot transmit unless lesions are

present on breast.

Pathogenesis : 56,57

Natural infection occurs only in the humans.T. Pallidum enters the body

through minor breaks in the skin and mucosa. After entering the body it multiplies

slowly at the site. However animal studies have shown that they appears in blood and

lymphatics within minutes and disseminate within hours.

Attachment to the host tissues may be by specific legands such as fibronectin,

laminin, collagen IV, collagen I and hyaluronic acid. From initial site it reaches blood

and lymphatics by producing hyaluronidase enzyme which degrades hyaluronic acid,

thus splitting the endothelial cell junctions.

Glycosaminoglycan on the outer surface of the organism and surface

associated sialic acid inhibits complement activation. It also synthesizes prostaglandin

E2 that down regulates early immune response.

Clinical manifestation :

Incubation period is 3-90 days and generation time 30-33 hours. The 50%

infection dose was calculated to be 57 organism. Clinical feature fall under following

stages.

45
1) Primary syphilis ;

Hunterian / hard chancre appears at the site of inoculation. It is a single

painless, indurated, avascular ulcer, round to oval, clearly defined with rolled borders,

covered by thick hemorrhagic exudates very rich in spirochetes. Multiple chancres

may be seen in immunodeficient persons. Most commonly seen in genital region,

rarely on mouth, nipples or fingers. The regional lymphnodes are swollen painless,

firm and descrete. Patient is highly infectious. It heals even without treatment in 3-6

weeks leaving a thin scar.50

Secondary syphilis :

It begins by 2-8 weeks after primary lesion heals. It is the most infectious

stage due to wide spread multiplication and dessimination. There is wide spread rash

(macular / maculopapular / pustular)

Characteristically involves palms, soles, oropharynx, in most interginous

areas, moist grey white plaque called condylomata lata are seen. Systemic symptoms

may be present. Any organ may be involved. They usually undergo spontaneous

resolution taking as long as 3-5 years.

Latent syphilis :

The disease becomes subclinical, diagnosis is possible only by serological

tests. It is of indefinite duration. This may be followed by natural cure in many cases.

But in few cases tertiary syphilis can occur after several years.

Tertiary / Late syphilis ;

It consists of cardiovascular lesions, gammata and meningovascular lesions. It

is because of delayed hypersensitivity and very few sprochetes are present.

46
Quarternary syphilis :

Neurological manifestation may develop in few cases after many decades.51

Congenital syphilis :56

Transplacental infection is most likely to occur during primary and secondary

stages. It may infects fetus at any time but the likelihood of clinical disease increases

as the pregnancy progresses. Many of the infected fetus die. Of those who survive half

are asymptomatic and the remaining develop lesions of secondary syphilis without

primary lesions. Hepatosplenomegaly, meningitis, anaemia, thrombocytopenia, bone

and teeth abnormalities can develop.

Laboratory diagnosis :

T. palladum is not easily cultivable or stainable bacteria. However gold

standard for diagnosis is invivo culture by intratesticular inoculation of rabbits. But it

is time consuming and expensive. So not routinely used.

Various diagnostic tests are :

1) Direct microscopic identification of T. pallidum

2) Serological tests to detect antibodies

3) Direct antigen detection test

1) Direct microscopic identification :51

1) Dark field microscopy :

This method is most specific and easiest method for diagnosis during early

syphilis before the appearance of antibodies.

Wet film of the exudates is examined under dark field microscopy. T pallidum

is identified by its typical microscopy and characteristic cork screw movements.

47
Direct fluorescent antibody test ;

It is more specific and more sensitive than dark field microscopy. This test

uses fluorescent dye tagged anti-T pallidum serum for staining the smear after fixing.

This can also be used to examine the tissue sections. Recently specific monoclonal

antibody is used to increase the specificity. To see under light microscope the smears /

tissue sections can be stained by silver impregnation methods (Fontana method /

levaditi method).

II) Serological tests :28,46,56,58

 Non treponemal tests : Nonspecific test that react with cardrolipin

 Treponemal tests : detects specific antibodies anti T.pallidum IgG, IgM and

IgA.

 Non treponemal tests are inexpensive, have high sensitivity and high negative

predictive value therefore used for screening.

 Treponemal tests are more specific with high positive predictive value,

therefore used for confirmation

Nontrepo nemal tests :

Also called as Reagin antibody tests are standard tests for syphilis these tests

uses cardiolipin antigen that reacts with reagin antibody (IgG).

Two most commonly used tests are Veneral research laboratory (VDRL) and

rapid plasma reagin card test. Non specific tests include :

 Wasserman CF reaction (1906)

 Kahn flocculation test (1928)

 Venereal disease research laboratory ( VDRL) test ( 1946)

 Rapid plasma reagin test (RPR),1957.

48
  Automated RPR test (1968)

 VDRL ELISA test (1987)

Initially donor resting was undertaken using nontrepenemal test such as RPR

test. These test though nonspecific have advantage of identifying primarily active or

recent infection.

VDRL test : It is a slide flocculation test that uses a purified lipid extract of beef

heart (cardiolipin) with added lecithin and cholesterol as standardized by Paugborn

(1945). Inactivated serum is mixed with cardiolipin antigen and kept on VDRL rotator

for 4 min. Cardolipin reacts with reagin antibody to form visible clumps which is read

under a low power microscope. Results are given as reactive, weakly reactive or non

reactive for quantitative reporting serial dilution of the serum to be used.

Rapid plasma re agin test :

It uses VDRL antigen containing fine carbon particles which makes the result

clear to the naked eye and it can be done on untreated serum or plasma but not

suitable for CSF.

Non trepenemal test are of 70-80% sensitive in primary stage and 100%

sensitive in secondary stage. After secondary stage titre diminishes. The test becomes

negative after 6-18 months of treatment so useful for monitoring the treatment.

It is commonly used for donor screening.

Teponemal tests :

These tests are mainly used for confirmatio n and are not suitable for routine

screening as they have lengthy procedure and need bound man power.

49
 In these tests entire T pallidum or its fragments are used as antigen to detect

specific antibodies (IgG) against treponemal cellular components. These test are used

to verify non-treponemal tests.

 Teponema pallidum immobilization (TPI) test (1949)

 Flourescent treponemal antibody absorption (FTA-ABS) (1964)

 T. Pallidum Haemagglutination assay (TPHA) (1967)

 T. Pallidum enzyme immune assay (TP-EIA) 1985, 1988.

TPI test : Is based on the ability of patients antibody and comple ment to immobilize

treponemes (N ichol’s strain) as observed by dark field microscopy. It is complicate

difficult, expensive and time consuming. So not in use now.

FTA-ABS test : Is indirect immunofluorescence test where Nichol’s strain fixed on

glass slides is used. The patient serum is first diluted in sorbent (reiter) treponema to

remove non specific treponemal antibodies. The serum is added to the slides. If it

contains antibody it coat the treponemes. Now add fluoroscence isothiocyanate

labelled antihuman Ig to the slide and examine under fluorescent microscope.It is first

to become positive in early syphilis and has higher sensitivity than non-treponemal

tests in late syphilis. However it can be done only in suitably equipped laboratories.

TPHA : Test uses tanned erythrocytes sensitive with T.pallidum as antigen. It is as

specific as FTA – ABS test except in primary stage. It is much simpler and

economical and tests are available commercially. Hence TPHA is used as standard

confirmatory test in many labs.

EIA : Have been developed using T.pallidum. antigens. These are available

commercially.

50
 Specific treponemal tests are of no value in monitoring the treatment as they

remain positive in spite of treatment.

Detection of IgM antibodies can be used as a indicator of active syphilis for

diagnosis of congenital syphilis and reinfection. It appears by 2 week of infection and

disappears within 6 mont hs.

MALARIA :

Malaria is a protozoan disease transmitted by the bite of infected Anophe les

mosquitoes.

Four species of genus plasmodium cause nearly all infections in humans.

These are P.falciparum, P.vivax, P.ovale and P.malariae.59

Prevalence :

World wide there are an estimated 247 million malaria cases among 3 billion

at risk in 2006, causing one million deaths mostly of children under 5 years of age60

In India although the malaria incidence has now reduced to 1.82 million cases

from about 72 million cases in 1950s, it continues to be causes of concern.61

Transmission of malaria can occur after transfusion of whole blood, red blood

cells, white blood cells and platelets. Plasma fraction do not trans mit the disease. The

parasites of all species remain viable for one week in stored blood at 2-6 0C. However

plasmodium falciparum remain viable for 2-3 weeks.21,25

The first case of transfusion malaria was reported in 1911. 25 The risk of TT

malaria differs widely between low endemic countries where the infection is imported

51
frorm out side and regions of high prevalence of plasmodium infection in general

population.62

The risk of transfusion transmitted malaria in low endemic areas like US and

Europe is low, with only one in 3-4 million units transfused being potentially

infectious.62

Post transfusion malaria is particularly dangerous in pregnant women and

children.

Proper scrutiny of the donors and screening of the blood for malaria should be

done to prevent the transfusion transmitted malaria.25

Life cycle and pathogensis :59,63,64

Plasmodium vivax, P. ova le and P.malariae cause low levels of parasitaemia.

mild anaemia, and in rare instances splenic rupture and nephritic syndrome.

P.falciparum causes high levels of parasitemia, severe anaemia, cerebral

symptoms, renal failure, pulmonary oedema and death.

The life cycles of the plasmodium species are similar although P. falciparum

differs in ways that contribute to its greater virulence.

The infectious stage of malaria, the spirozoite is found in the salivary glands

of female mosquitoes. When the mosquito takes a blood meal spirozoites are released

into the humans’s blood and within minutes attach to and invade liver cells by binding

to the hepatocyte receptor for the proteins thrombospondin and properdin. Within

liver cells malaria parasites multiply rapidly, releasing as many as 30,000 merozoites

(asexual haploid forms) when each infected hepatocyte ruptures. P.vivax and P.ovale

52
form latent hypnozoites in hepatocytes which cause relapse of malaria long after

initial infection.

Once released from the liver, plasmodium merozoites bind by a parasite lectin

like molecule to sialic acid residues on glycopherin molecules on the surface of red

cells. Within the red cells the parasite grow in membrane bound digestive vacuole

hydrolyzing hemoglobin through secreted enzymes. The tropozoite is the first stage of

the parasite in the red cells and is defined by the presence of single chroma tin mass.

The next stage schizont has multiple chromatin masses each of which develops into

merozoite. On lysis of the red cells the new merozites infect additional red cells.

Although most malaria parasites within the red cells develop into merozoites some

parasites develop into sexual forms called gametocytes that infect the mosquito when

it takes its blood meal.

Plasmodium falciparum causes more severe disease than the other

plasmodium species do. Several features of p.falciparum account for its greater

pathogenicity. P. falciparum is able to infect red cells of any age leading to high

parasite burdens and profound anaemia when compared to other species which infect

only young or old blood cells which are a smaller fraction of the red cells pool. P.

falciparum causes infected red cells to clump together (rosette) and stick to

endothelial cells living small blood vessels (sequestration) which block blood flow.

Several proteins including p.falcparum erythrocyte membrane protein I

(PfEMP1) form knobs on the surface of red cells. PfEMP1 binds to ligands on the

endothelial cells including CD36, thrombospondin, VCAM-I, ICAM-1 and E-selectin.

Ischemia due to poor perfusion causes manifestation of cerebral malaria which

is the main cause of death due to malaria in children.

53
 P.falciparum stimulates production of high levels of cytokines including TNF,

IFN and IL-1. GPI linked proteins including meroozoite surface antigens are

released from infected red cells and reduce cytokine production by host cells by a

mechanism that is not yet understood. These cytokines suppress production of red

cells, increase fever, stimulate with nitric oxide production and induce expression of

endothelial receptor for PfEMP1.

Host resistance to plasmodium :64

There are two general mechanism of host resistance to plasmodium.

First, inherited alterations in red cells make people resistant to plasmodium. People

who are heterozygous for sickle cell trait, HbC and Buffy negative individuals are

resistant to infection.

Second : Repeated or prolonged exposure to plasmodium species stimulate an

immune response that reduces the severity of the illness caused by malaria.

Clinical features :38,59

Malaria is a very common cause of fever in tropical countries.

The first symptom of malaria are non specific : lack of a sense of well being,

headache, fatigue, abdominal discomfort and muscle aches followed by fever, nausea,

vomiting and orthostatic hypotension are common.

Fever : Fever occurs in characteristic paroxysms in which fever spikes, chills and

rigors occur at regular interval. Though relatively unusual with other species, such

pattern of fever suggests infection with P.vivax and P.ovale.

54
Febrile convulsion may occur with any type of malaria but generalized seizures are

specifically associated with falciparum malaria and may herald the onset of cerebral

malaria.

The majority of the symtoms are caused by release of merozoites into the

blood stream. The anemia resulting from destruction of the red cells and problems

caused by large amount of free hemoglobin released into the circulation after red

blood cells rupture.

Transfusion malaria :59

Malaria can be transmitted by blood transfusion, needle stick injury, sharing of

needle by infected injection drug users or organ transplantation. The incubation period

in these setting is often short because there is no pre-erythrocytic stage of

development.

Diagnosis : 59,63,65,66

The most important aspect is clinical diagnosis

1) Parasitological methods :

The only of certain means of diagnosing all four species of human malaria is

the detection of the plasmodium species by microscopic examination of the blood.

Thick film : The thick film method concentrates layers of red blood cells on small

surface by a factor of 20-30, is the most sensitive and by for the best for clinical use.

Thin film : Provide clues regarding the species and degree of disease severity, which

includes high level parasitema, mature ring stage parasites or high number of

circulatory schizonts.

55
Acridine stained film : staining with acridine orange, which is read either on a

fluoroscope or a microscope equipped with an interference filter system allows

quicker screening of films because parasites are more readily recognized and a lower

power microscope objective may be used.

QBC method : Also based on acridine orange staining. The blood is centrifuged in a

specially designed and patented micropapillary tube fitted with a plastic float. The

float spread the buffy coat against the edge of the tube. Parasites and leukocytes take

up the dye which is fluorescent when examined under ultraviolet light. This elegant

method is easy to perform, fasts and easy to read, but require specialized equipment

and the expensive QBC capillary tubes. The sensitivity of QBC when used in field

conditions is comparable or marginally better than thick films.

Immunological methods :

Immunological methods provide the means for detecting either the parasite

antigen or the host antibodies directed against the parasite.

Detection of antigens is an acceptable alternative to parasite detection.

Detection of antibodies merely provide information on past malaria experience

and is of limited use for individual diagnosis.

SEROLOGY : Methods in use since 1960.

1) IFAT : Main method for routine serodiagnosis. Relatively easy to make antigen

slides for all human malarial parasites. The disadvantage is it needs fluoroscope.

2) Indirect hemogglutination assays

3) ELISA : Uses soluble malaria antigen, prepared from asexual stages of

P.falciparum which can be readily grown in vit ro coated on the walls of microtiter

56
 plate. A large samples can thus be processed at the same time and produce

quantitative results when the assay is read on spectrophotometer. ELISA has been

applied to variety of defined, synthetic or recombinant malarial antigens.

Other serological tests includes radioimmunoassay latex agglutination, indirect

hemagglutination, solid phase dipstick and membrane dot blot which may have

specific advantage in given situation.

ANTIGEN DETECTION :

In contrast to serology, positive antigen detection assay should only detect

current infection tests for detecting malarial antigen are based on either an antigen

capture or an antigen competetion format and often use ELISA or radio immunoassay

(RIA) methodology.

The best antigen assay described have a maximum sensitivity of 0.01 to 0.001

percent parasitaemia and are five to ten times inferior to good quality microscope.

Parasigt – F first commercial detection test in dipstic format in which monoclonal

antibody captures a specific antigen of p.falciparum; PfHRP-2 present in the parasite

throughout the erythrocytic cycle.

If antigen is present,the positivity of the test is visualized by a second anti

HRP2 antibody labelled with a color marker which produce a visible line on the

dipstick while test take only 10mins and sensitivity almost comparable to thick film.

Immunochromatographic rapid malaria strip :

Principle : Based on detection of parasite lactate dehydrogenase the PLDH has

different biochemical characteristics from human lactate dehydrogenase Therefore

differentially measured using a simple calorimeter assay

57
OptiMal : Principle of optiM al based on detection of PLDH using series of

monoclonal and polyclonal antibodies : Recently developed immunochromatographic

rapid malaria strip test.

Differentiation of malaria species in the optiMal test is based on the antigen

differences between PLDH isoform. Unless HRP-2 PLDH doesnot persists in the

blood but clears at about the same time as the parasites. Following successful

treatment, test is useful for monitoring the therapy and detecting drug resistant

malaria,. because the level of PLDH correlates with number of viable malaria

parasites in the blood. Test is sensitive and able to distinguish between P.vivax and

P.falciparum.

Molecular methods :

It is used in research tool:

Principle : Based on the principle of nucleic acid hybridization in order to detect

parasite DNA or RNA.

A known sequence of nucleic acid is (oligonucleotide) is synthesized and

labeled either with radio active P or non radioactive calorimetric reagent and this

probe is used to detect parasites nucleic acid, taking advantage of the fact that

complementary sequences will hybridize.

PCR : technique capable of detecting parasitemia of less than 0.00002 percent.

58
Methodology
 METHODOLOGY

The present study was carried out in Blood bank of J.J.M Medical College,

Davangere from July 2009 to June 2011.

The blood bank of department of pathology, J.J.M. Medical College is

licenced blood bank with average annual collection of 8000 units of blood from

healthy blood donors from in and around Davangere annually.

During the study blood units were screened for HIV, HBsAg, HCV, syphilis

and malaria.

The blood units were collected from voluntary and replacement donors. A

voluntary donor is one who donates voluntarily and is not paid for it. A replacement

donor is nonremunerated donor who donates blood for a particular patient admitted in

hospital.

Methods employed :

Donor selection :

The donors were selected by detailed history and physical examination

according to the criteria laid down in the standard operating procedure of our blood

bank. Blood sample were collected from all healthy male and female donors. The

selected donors were subjected to phlebotomy.

Sample collection :

Two ml of blood sample was collected in labelled pilot tube at the time of

collection of blood from donor tubing of blood bag the sample was further centrifuged

at 3500 rpm for 5 minutes to obtain clear non hemolyzed serum. The samples were

59
tested for HIV, HBsAg, HCV, syp hilis. Malaria was screened by thick and thin blood

smears.

Inclusion criteria: Healthy voluntary and replacement donors

Exclusion criteria: Blood donors who are unfit to donate blood according to standard

blood donors criteria

I) Screening test for HIV :

ELISA :

SD HIV 1/2 ELISA – 3.0 third generation indirect ELISA

kit (Biostandard diagnostic Pvt. Ltd. India).

Principle :

The kit contains a microplate which is precoated with recombinent HIV 1/2

antigens (gp41, p24 and gp36 on microtitre wells. Specimen and controls are added to

the microtitre wells and incubated. Antibodies to HIV 1 / 2 if present in the specimen

will bind to the specific antigens absorbed on the surface of the wells. The plate is

then washed to remove unbound material. The recombinant HIV 1/2 antigens

enzymes conjugate is bound to anti HIV 1/ 2 by forming a “sandwich”. All unbound

materials are removed by washing. Finally substrate solution containing chromogen

and hydrogen peroxide is added to well and incubated. Color develops in proportion

to the amount of HIV antibodies present in the specimen. The enzyme substrate

reaction is read by ELISA reader for absorbance at the wavelength of 450nm.

Kit presentation : 96 test pack.

60
Kit and its components of SD HIV 1 /2 ELISA 3.0.

Material provided :

Material
Remark 96T/kit
reagent
Coated 96 wells, coated with recombinant HIV1- gp41 1 plate
microplate (including subtype O), p24 HIV-2, gp36 antigen. Keep
unused wells at 2-80C in the provided aluminium bag and
accurately sealed
Enzyme Recombinant HIV-1, gp 41 (including subtype O), p24 1 vial
conjugate HIV-2, gp36 antigen conjugated to horseradish (20ml)
peroxidase (HRPO).
Preservation : Procilin 300 (0.05%). Ready for use.
Sample Phosphate buffer, bovine serum, and stabilizer, ready for 1 vial
diluent use. Preservative : sodium azide (0.5ml)
Negative Normal human serum. Preservative ; sodium azide 1 vial
control (0.5ml)
TMB Sodium acetate, hydrogen peroxide and gentamicin, store 1 vial
Substrate A unused TMB substrate A at 2-80C (10ml)
Note – Before using make 1:1 mix with TMB substrate B
TMB Tetramethyl benzidine (TMB) hydrochloric acid store
substrate B unused TMB substrate B at 2-80C
Note : Before using make 1:1 mix with TMD substrate A
Washing PBS – Tween 20 preservative : Procline 300 (0.05%) 1 vial
solution Note : Before use, take 50ml of wash concentrate and (50ml)
then fill up to 1,000 ml with distilled water. In presence
of undissolved crystals resuspend the solution by placing
the vial at 370C few minutes
Stopping 1.6 N sulfuric acid ready for use 1 vial
solution (20ml)

61
Procedure :

1) Prepare the strip wells for, negative control 3 wells, positive control 2 wells and

sample wells.

2) Pipette 100 l of sample diluent to each well.

3) Add 50 l negative control to 3 wells, positive control to 2 wells, and sample to

each well.

4) Cover the microplate with adhesive plate sealer and mix well on vibrating mixer.

Mixing is very important to get the reproducible result.

5) Incubate the wells at 370C for 30 min.

6) Wash the well 5 times with 350l of diluted washing solution giving at least 10

second soak time for each wash and aspirate all liquid from the wells.

7) Pipette 100l conjugate to each well.

8) Cover the microplate with adhesive plate sealer

9) Incubate the wells at 370C for 30 minutes.

10) Wash the well 5 times with 350l of dilute washing solution giving at least 10

seconds soak time for each wash and aspirate all fluid from the wells.

11) Gently mix the TMB substrate A & B at the ratio of 1:1 and pipette 100l of

mixed substrate solution to each well.

12) Incubate the wells for 10 minutes at room temperature.

13) Pipette 100l of stopping solution to each well.

14) Read the absorbance of the wells with bichromatic spectrometer at 450nm.

Reading must be completed within 1 hour from the end of assay.

62
Interpretation of results

1) Test validation : The individual values of the absorbance for the control sera

are used to calculate the mean value.

If 0.010  absorbance (Neg)  0.200

0.010  absorbance (Pos)  1.000

If these specification are not met the test to be repeated.

2) Evaluation : Calculate the mean absorbance of the negative controls, then

calculate the cut off value by adding 0.300.

Cut off value = mean absorbance (neg) + 0.300

Test results :

1) Absorbance (sample) < cut off value = negative

2) Absorbance (sample) > cut off value = positive

Test specimen with absorbance value within 10% below the cutoff should be

considered suspect and should be repeated in duplicate.

II) Screening test for hepatitis B virus (

HBsAg) ELISA

SD HBsAg ELISA 3.0 – Enzyme immunoassay for detecting HBsAg

(Biostandard diagnostics pvt. Ltd, India)

Principle :

SD HBsAg ELISA 3.0 is a enzyme linked immunosorbent assay for the

qualitative detection of hepatitis B virus surface antigen (HBsAg) in human serum or

plasma).

SD BsAg ELISA 3.0 contains a microplate, which is precoated with anti-

hepatitis surface antigen (anti-HBs) on well. During first incubation HBsAg in patient

63
serum is bound to anti-HBS on well and is bound to anti- HBS enzyme conjugate, by

forming a “sand wich”. Following this incubation all unbound material are removed

by aspiration and washing the residual enzyme activity bound in the wells will thus be

directly proportional to the HBsAg concentration in patient serum and evidenced by

incubating the solid phase with substrate solution (TMB) in a substrate buffer.

Calorimetric reading will be performed by using a spectrophotometer at 450nm.

SD HBsAg ELISA 3-0 is double sandwich ELISA for the qualitative detection of

HBsAg with high degr ee of sensitivity and specificity.

Materials provided :

Material Remark 96 test kit

reagent
Coated 96 wells, coated with anti-HBsAg (anti HBS). Keep 1 plate
microplate unused wells at 2-80C in the provided aluminium bag and
accurately sealed
Enzyme Anti-HBS conjugated to horse radish peroxidase (HRPO). 1 vial
conjugate Preservative : proclin 300 (q.s.). Ready for use (5ml)
Positive HBsAg positive human serum. Preservative : proclin 300 1 vial
control (q.s) (2ml)
Negative Normal human serum Preservative: Proclin 300 (q.s) 1 vial
control (2ml)
TMB Sodium acetate, hydrogen peroxidase and gentamicin 1 vial
substrate A stored unused TMB substrate A at 2-80C (10ml)
Note : Before using make 1:1 mix with TMB substrate B
TMB Tetramethyl benzidine (TMB) hydrochloric acid 1 vial
substrate B Stored unused TMB substrate B at 2-8 C. Note before
0
(10ml)
using make 1:1 mix with TBM substrate A.
Washing PBS – Tween 20 Note – before use, take 25ml of wash
solution concentrate, and then fill- up to 500ml with dis tilled water
in presence of undisolved crystals, re-suspend the solution

64
 by placing the vial at 370C for few minutes.
Stopping 1.6 N sulfuric acid 1 vial
solution Ready for use (20ml)
Adhesive
plate sealer

Procedure :

1. Prepar the test strip wells and other components and place it at room temperature.

2. Prepare the strip wells for negative control 3 wells, positive control 2 wells, and

sample to each well.

3. Add 100l of negative control to 3 wells, positive control to 2 wells and sample to

each well.

4. Pipette 25l of enzyme conjugate to each well.

5. Cover the microplate with adhesive plate sealer and mix well on vibrating mixer.

Mixing is very important to get the reproducible results.

6. Incubate the wells at 37  10C for 60 minutes.

7. Wash the wells 5 times with 350 l of dilute washing solution. giving at least 10

seconds soak time for each wash and aspirate all liquids from the wells.

8. Gently mix the TMB substrate A and B at the ratio of 1 to 1 and pipette 100 l of

mixed substrate solution to each well.

9. Incubate the wells for 10 minutes at room temperature.

10. Pipette 100 l of stopping solution to each well.

11. Read absorbance of the wells with bichromatic spectrometer at 450nm with

reference wavelength of 620nm. Reading must be completed with 30 minutes

from the end of the assay.

65
Interpretation of results :

1. Test validation

The individual values of the absorbance for the control sera are used to

calculate the mean value if - 0.005  A (neg)  0.200

A (pos)  1.000

If one of the absorbance values of negative control is outside the specification

this value can be neglected.

Both absorbance values of the positive controls must comply with

specification.

If these specifications are not met, the test is to be repeated.

2. Evaluation :

Calc ulate the mean absorbance of the negative controls then calculate the cut-

off value by adding 0.050

A (neg) + 0.050 = cut off value

Test results

i) A (sample)  cut off : HBs Ag negative

ii) A (sample)  cut off value : HBsAg positive.

Samples with a test result which is equal to or greater than the cut-off value should

first be tested in duplicate. If in the retest the mean absorbance is again equal or

greater than the cut-off, such samples should be verified using a confirmatory test.

66
3) Screening test for Hepatitis C Virus.

ELISA :

SD HCV ELISA 3.0

Principle :

SD HCV ELISA 3.0 contains a microbplate which is precoated with

recombinant HCV antigens. (Core, NS3, NS4, NS5) on well. During first incubation,

anti-HCV in patient serum is bound to the recombinant HCV antigens. Following this

incubation, all unbound material are removed by aspiration and washing. The

antihuman IgG peroxidase-enzyme conjugate is bound to anti HCV. Following the

incubation all unbound material are removed by aspiration and washing. The residual

enzyme activity found in the wells will thus directly proportional to the anti HCV

concentration in patient serum and evidenced by incubating the solid-phase with

substrate solution in substrate buffer. Colorimeter reading will be performed by using

a sphectrometer at 450nm. SD HCV ELISA 3.0 is indirect sandwich Elisa for the

qualitative detection of antibodies against HCV.

Materials provided
Material / Remark 96T/kit
reagent
Coated 96 wells, coated with recombinant HCV core, NS3, NS4, 1 plate
conjugate NS5 antigen. Keep unused wells at 2-80C in the provided
aluminium bag and accurately sealed.
Enzyme Goat antihuman IgG conjugate to horseradish peroxidase 1 vial
conjugate (HRPO) : Preservative : Proclin 300 (0.05%) ready for (20ml)
use
Sample diluent Phosphate buffer, bovine serum, and stabilizer. Ready 1 vial
for use. Preservative : Proclin 300 (0.05%) (20 ml)
Positive Anti HCV positive human serum. Preservative : Proclin 1 vial
control 300 (0.05%) (0.5ml)

67
 Negative Normal human serum. Preservative: Proclin 1 vial
control (0.5ml)
TMB substrate Sodium acetate, hydrogen peroxide and gentamicin. 1 vial
A Store unused TMB substrate A at 2.80C. Note before (10ml)
using make 1:1 mix with TMB substrate B
TMB substrate Tetramethyl benzidine (TMB) hydrochloric acid 1 vial
B Stored unused TMB substrate B at 2-80C (10ml)
Note before using make 1:1 mix with TBM substrate A
Washing PBS – Tween 20 Preservative : Proclin 300 (0.05%) 1 vial
solution (20 x Note : Before use take 50ml of wash concentrate. and (50 ml)
concentrated) then fill up to 1000ml with distilled water. In presence of
undissolved crystals, resuspend the solution by placing
the vial at 370C for few minutes.
Stopping 1.0 N sulfuric acid, ready for use 1 vial
solution Adhesive plate sealer (20ml)

Procedure :

1. Prepare the strip wells for negative control 3 wells, positive control 2 wells,and

samples.

2. Pipette 100l of sample diluent.

3. Add 10l of negative control 3 wells, positive control 2 wells and sample each

well.

4. Cover the microplate with adhesive sheet and mix well on vibrating mixer. Mixer

is very important to get the reproducible results.

5. Incubate the wells at 370c for30 minutes.

6. Wash the well with 350l of dilute washing solution giving at least 10 seconds

soak time for each wash and aspirate all liquid from the wells. Strict adherence to

the washing procedure is important to ensure optimum assay performance. Follow

the specific soak time, 350l of washing volume and 5 times of washing.

68
7. Pipette 100l enzyme conjugate each well.

8. Cover the microplate with adhesive sheet

9. Incubate the wells for 30 minutes at 370C

10. Wash the wells 5 times with 350l of diluted washing solution giving at least 10

seconds soak time for each wash and aspirate all liquid from the wells.

Strict adherence to washing procedure is important to ensure optimum assay

performance.

11. Gently mix the TMB substrate A and B at the ratio of 1:1 and pipette 100l of

mixed substrate solution to each well.

12. Incubate the well for 10 minutes at room temperature.

13. Pipette 100l of stopping solution to each well

14. Read the absorbance of the well with bichromate sphectrometer at 450nm with

reference wavelength at 620nm. Reading must be completed within 1 hr from the

end of the assay.

Interpretation of results :

1. Test validation

The individual values of the absorbance for the control sera are used to

calculate the mean value if

- 0.010  A (neg)  0.200

A (pos) 1.000

If one of the absorbance values of negative controls in outside the

specification this value can be neglected.

Both absorbance values of the positive control must comply with the

specification. If these specification are not met the test is to be repeated.

69
2. Evaluation :

Calculate the mean absorbance of the negative controls then calculate the

cutoff value by adding 0.400

A (neg) + 0.400 = cut off value.

Based on the criteria of the test, the samples are classified as follows

: Test results :

1. A (sample) = < cutoff = anti HC V neg.

2. A (sample)  cutoff  anti HCV positive.

Screening test for syphilis

Rapid Plasma Reagin test kit (BEACON diagnostic Pvt. Ltd)

Principle :

RPR antigen suspension is a carbon containing cardiolipin antigen, which

detects “reagin” antibody present in serum of persons with syphilis. When a specimen

contains antibody, flocculation occurs due to coagulation of the carbon particles of the

RPR antigen which appear as black clumps against the white back ground of the card.

This coagulation is read manoscopically. Non reactive specimens show an even light

gray color.

Requirements for the test

1. Serum sample

2. Reagents in kit

Reagent 1 : RPR antigen

Reagent 2 : Positive control

Reagent 3 : Negative control

70
 3. Accessories

Plastic droppers

Mixing sticks

Rubber teat

Delivery dropper

Plastic slides

Procedure (As per manufacturers instructions)

1. Place one drop of serum/plasma (50l) on the slide with disposable serum

dropper.

2. After gently mixing RPR antigen suspension place one drop (15-20l) by

antigen dropper.

3. Mix well and spread out the liquid on entire area of circle by using disposable

mixing stick

4. Rock the slide gently for 6 minutes and observe under good light source for

appearance of carbon particles clumping.

Interpretation of result :

Positive result: Black aggregates (carbon) which may be deposited at the periphery.

The liquid appearing before 4 minutes of rotation

Read the results under strong source light with a hand lens. Test results

showing slight but definite clumping is reported as reactive or positive.

Negative result :

Complete absence of black aggregates with uniform grayish background at the

end of 4th minute rotation.

71
Screening test for malaria :

Microscopic examination of Leishman stained thick and thin blood smears

was used as a screening test for malaria.

Preparation of smears :

Thin smear : This smear is prepared on a clean, grease free slide by spreading a

small drop of blood evenly on the slide. So that there is only one continuous layer of

cells.

Thick smear : Thick smear is prepared by placing 4 drops of blood in the centre of

slide and spreading it out with a corner of another slide to cover an area about 4 times

its original area, to an even thickness so that with a slide placed in piece of news

paper, small print is just visible. Allow the film to dry thoroughly for at least 30

minutes at 370C. After drying thick film is dehemoglobinized with water before

staining.

Leishman’s staining of thin and thick smears :

1. Airdry the films and flood the slide with the stain.

2. After 2 minutes add double the volume of water and stain the film for 5-7

minutes.

3. Then wash it in a stream of buffered water until it has acquired a pinkish ting

(upto 2min)

4. After the back of the slide has been wiped clean set it upright to dry.

72
Microscopic examination of smear of or malarial parasite.

After screening the smear on 40X object for protozoan, 100X oil immersion

objective is used to detect plasmosidium species.

At least 100 oil immersion fields of thick film and 200 oil immersion fields of

thin film using 100x objective are exa mined before labeling the blood unit as

negative for parasite.

Statistical analysis: prevalence of each transfusion transmissible infection

was compared between voluntary and replacement donors by statistical analysis.

Z test for proportion was used to compare between two donor categories

73
 Fig.6 : ELISA Reader

Fig. 7 : ELISA instruments

(A) Kit components
(B) Plate washer (LISA wash)
(C) Micro plate Reader (BIO RAD)

74
Fig. 8 : ELISA Kits

75
Results
 RESULTS

The present study was carried out in Blood Bank, Department of pathology of

J.J.M Medical College Davangere, during the period from July 2009 to June 2011.

During the study total 16,872 donors blood units were screened for HIV, HBsAg,

HCV, Syphilis and Malaria. The donor age ranged from 18-60 yrs, majority (76.2%)

in the age group of 18-35 yrs.

Out of the 16,872 blood donors, 15,456 (91.6%) were replacement donors

and remaining 1,416 (8.4%) were voluntary donors.

Table No.1: Showing Type of blood donors

Type of donor No. of screened blood units Percentage

Voluntary 01,416 8.4%

Replacement 15,456 91.6%

Total 16,872 100%

Graph 1: Pie chart showing type of donors

76
Sex distribution; Out of the total 16,872 donors, males constituted 16,451(97.5%)

and only 471 (2.5%) donors were females.

Table No.2: Sex wise distribution of blood donors

Sex No of screened blood units Percentage

Male 16,451 97.5%
Female 0471 2.5%
Total 16,872 100%

Graph 2: Pie chart showing sex distribution of donors

Age distribution : Maximum donors were between the age group of 18-35 years

constituting 76.21%.

Table 3 : Age distribution of blood donors :

Age in years No. of donors Percentage

18-25 6329 37.51%
26-35 6530 38.70%
36-45 2517 14.92%
46 and above 1496 8.87%
Total 16872 100%

77
SEROPREVALENCE OF TTI :

Out of the total 16872 screened blood units 312 units were seropositive for

transfusion transmissible infections (TTI), giving prevalence rate of 1.85%. Out of

this 305 were replacement donors and remaining 7 were voluntary donors.

Table No.4: Seroprevalence of TTI in total donors .

Total No.donors No.of positive donors Seroprositivity (%)

16,872 312 1.85%

Table No .5: Seroprvalence in voluntary and replacement donors.

Type of donors No. of donors No. of positives Seripositivity (%)

Voluntary 01,416 07 0.49%

Replacement 15,456 305 1.96%

Total 16,872 312 1.85%

Graph 3 : Seropositivity in different types of donors / total donors

1.96
2 1.85
1.8
1.6
1.4
1.2
Seripositivity

1
0.8
0.6
0.49
0.4
0.2
0

Voluntary Replacement Total

Type of Donors

78
Table 6 : Seroprevalence of TTI

HIV HbsAg HCV Syphilis

Donors No.
n (%) n (%) n (%) n (%)

Voluntary 1416 00 (0%) 07 (0.49%) 00 (0%) 00 (0%)

Replacement 15456 28 (0.18) 260 (1.68) 15 (0.10%) 02 (0.02%)

Total 16872 28 (0.17) 267 (1.58%) 15 (0.09%) 02 (0.01%)

Graph 4 : Seroprevalence of TTI

1.8 1.68
1.6 1.58
1.4
1.2
1
0.8
0.6
Percenta

0.4 Voluntary donors Replacement donors

0.2 Total donors
0 0.49

0.180.17
0.100.09
0 0.020.01
0 0
Syphilis
HIVHbsAgHCV
Donors

The overall seroprevalence for HIV, HBsAg, HCV and syphilis was 0.17,

1.58, 0.09 and 0.01 respectively. No blood donors were positive for malaria parasites.

79
SEROPREVALENCE OF HIV IN BLOOD DONORS

Among the 16,872 total donors, the HIV positive donors were 28, with a

seroprevalence of 0.17%.

Table No. 7: Seroprevalence of HIV in total donors

Total donors HIV positive units Seroprevalence

16872 28 0.17%

Table No. 8: Seroprevalence of HIV in different donor category

No of screened No. of seropositive

Donor category Percentage
blood units units

Voluntary 1,416 00 00

Replacement 15,456 28 0.18%

Total 16,872 28 0.17%

( Z = 0.09, p = 0.92 )

Graph 5 : HIV Seropositivity in different types of donors / total donors

0.2 0.18
0.2 0.17
0.2
0.1
0.1
Percenta

0.1
0.1
0.1
0.0
0.0
0.0 0

Voluntary Replacement Total

Donor category

80
 Above table shows that out of total 16,872 blood units screened 28 (0.17%)

units were seropositive for HIV. And all of the seropositive were replacement donors.

The percentage seropositivity in replacement donors is 0.18%. However the

difference was statistically not significant.

Age distribution : Out of the total 28 seropositive HIV donors majority (20) were in

the age group 18 to 35 years cases

Table 9: Age -wise distribution of HIV seropositive donors

Age range (yrs) No. of positive cases Percentage (%)

18-25 04 14%

26-35 18 65%

36-45 04 14%

46 and above 02 07%

Total 28 100%

Sex Distribution : All of the seropositive HIV donors were males.

Table 10: Sex wise distribution of HIV seropositive donors

Sex No. of seropositives Percentage

Male 28 100%

Female 00 -

Total 28 100%

81
Marital status : Out of the total 28 seropositive HIV donors 18 were married and 10

were unmarried.

Table 11: Marital status in HIV positive donors :

Marital status No.of seropositives Percentage

Married 18 64%

Unmarried 10 36%

Total 28 100%

Geographic distribution : Out of the 28 seropositive HIV donors 16 were from

urban and 12 were from rural areas.

Table 12 : Geographic distribution of HIV positive do nors

Area No.of seropositives Percentage

Urban 16 57%

Rural 12 43%

Total 28 100%

82
SEROPREVALENCE OF HBsAg IN BLOOD DONORS.

Out of the total 16,872 blood donors 267 (1.58%) were positive for HBsAg.

Out of 267 positive donors, 260 were replacement donors and 7 were voluntary

donors. The seroprevalence of HBsAg in total donors was 1.58%.

Table No.13: Seroprevalence of HBsAg in total donors

Total donors No. of positives Seroprevalence

16,872 267 1.58%

The seroprevalence of HBsAg among voluntary donors and replacement

donors was 0.49% and 1.68% respectively. However the difference was statistically

not significant

Table No. 14: Seroprevalence of HBsAg in different donor categories.

Donor category No of screened No. of seropositive Percentage

blood units units
Voluntary 01,416 07 0.49%

Replacement 15,456 260 1.68%

Total 16,872 267 1.58%

( Z=0.46, p=0.65)

Graph 6 : HBsAg Seropositivity in different types of donors / total donors

1.8 1.68
1.58
1.6
1.4
1.2
1
Percentag

0.8
0.6
0.4
0.2 0.49
0

Voluntary Replacement Total

Donor category

83
Age distribution: out of the total 267 HBsAg positive donors majorit y ( 223) were

in the age group of 18-35 years.

Table 15 : Age wise distribution of HBsAg positive donors

Age range (yrs) No. of positives Percentage

18-25 75 28%

26-35 148 56%

36-45 36 13%

46 and above 08 03%

Total 267 100%

Table 16 : Sex wise distribution of HBsAg positive donors

Sex No .of positives Percentage

Male 264 98.8%

Female 03 0.2%

Total 267 100%

Out of the total 267 seropositive donors for HBsAg, 264 were males and 3

were female donors

84
Marital status in HBsAg positive donors;

Out of the total 267, HBsAg positive donors 192 were married and remaining 75 were

unmarried.

Table No.17: Marital status in HBsAg positive donors

Marital status No . of positives Percentage

Married 192 71.9%

Unmarried 75 28.1%

Total 267 100%

Geographical distribution: out of the total 267 positive HBsAg cases 141(53.2%)

were from urban are and remaining 126 (46%)were from rural areas.

Table No.18: Geographical distribution of HBsAg positive cases.

Area No.of positives Percentage

Urban 141 53.2%

Rural 126 46.8%

Total 267 100%

85
SEROPREVALENCE OF HCV

Among 16872 total donors, 15 donors were reactive for HCV antibody, with a

seroprevalence of 0.09%

Table No. 19: Seroprevalence of HCV in total donors

Total donors No. of positives Seroprevalence

16,872 15 0.09%

Table No. 20: Seroprevalence of HCV in different donor categories.

No of screened No. of seropositive

Donor category Percentage
blood units units
Voluntary 01,416 00 00%

Replacement 15,456 15 0.10%

Total 16,872 15 0.09%

( z=0.17, p= 0.87 )
As the above table shows that out of the total 16,872 screened blood units 15

(0.09%) were seropositive for HCV. All the seropositive units were from replacement

donors. None of the blood units from voluntary donors were positive for HCV. The

seroprvalence in replacement donors was 0.10%. However the difference was

statistically not significant.

Graph 7 : HCV Seropositivity in different types of donors / total donors

0.1
0.1 0.09

0.08

0.06
Percenta

0.04

0.02
0
0
Voluntary Replacement Total
Donor category

86
Age distribution: Out of the total 15 HCV positive donors 10 were in the age group

between 18-35 years.

Table No.21: Age wise distribution of HCV positive donors

Age range ( yrs) No. of positives Percentage

18-25 4 26.66%

26-35 6 40.00%

36-45 3 20.00%

46 and above 2 13.34%

Total 15 100%

Sex wise distribution: All the 15, HCV positive donors were males. No female

donors were positive for HCV.

Table 22 : Sex wise distribution of HCV positive donors

Sex No. of positives Percentage

Male 15 100%

Female 00 00

Total 15 100%

87
Marital status: Out of the 15 HCV positive donors 12 (80%) were married and

3(20%) were unmarried.

Table 23 : Marital status in HCV positive donors

Marital status No. of positives Percentage

Married 12 80%

Unmarried 03 20%

Total 15 100%

Geographical distribution: Out of 15 donors positive for HCV 9 were from urban

area and remaining 6 were from rural area.

Table 24: Geographical distribution of HCV positive donors

Area No. of positives Percentage

Urban 9 60%

Rural 6 40%

Total 15 100%

88
SEROPREVALENCE OF SYPHILIS

Among the total 16,872 donors only 2 donors were positive for syphilis with a

seroprevalence of 0.01%

Table No. 25: Seroprevalence of syphilis

Total donors No. of positives Seroprevalence

16,872 02 0.01%

Table No.26: Seroprevalence of Syphilis in different donor categories.

No of screened No. of seropositive

Donor category Percentage
blood units units

Voluntary 01,416 00 00%

Replacement 15,456 02 0.02%

Total 16,872 02 0.01%

( z = 0.07, p = 0.84% )

Graph 8 : Syphilis Seropositivity in different types of donors / total donors

0.02
0.02
0.018
0.016
0.014
0.012
0.01 0.01
Percenta

0.008
0.006
0.004
0.002
0
0

Voluntary Replacement Total

Donor category

89
 As the above table shows that out of total 16,872 donors screened, 2 (0.01%)

units were seropositive for rapid plasma reagin test for syphilis. Two seropositive

units were from replacement donors. None of the blood units from voluntary donors

show seropositivity to RPR test for syphilis. However the difference was statistically

not significant.

Both the positive donors were males and one was married and other was

unmarried. One positive donor for syphilis was from urban and other one from rural

area.

Prevalence of Malaria.

Of the total 16,872 screened blood donors, none of the blood units were

positive for malaria parasites.

Co – infection:

Out of 16,872 total screened blood units, there was no co- infection of HIV

with HBsAg, HCV or syphilis. There was no co- infection of HBsAg with HCV

either.

90
Discussion
 DISCUSSION

The risk of transfusion transmissible infections (TTI) has declined

dramatically in developed nations over the past two decades, primarily because of

extraordinary success in preventing HIV and other established transfusion transmitted

viruses from entering the blood supply. But same may not hold good for the

developing countries. The National Policy for Blood Transfusion Services in our

country is of recent origin and the transfusion services are hospital based and

fragmented.67

The present study was carried out in the Blood Bank, Department of pathology

of our institute during the period from July 2009 to June 2011. During the study

period 16,872 blood units were screened for HIV, HBsAg, HCV, Syphilis and

Malaria.

The donors age ranged from 18-60 yrs. Similar age range was observed in

other studies. In our study 98.5% donors were males while only 2.5% donors were

females. This could be explained on the basis that Indian women have a very high

incidence of anaemia, especially in the child bearing age and hence are likely to face

disqualifiacation while being screened for blood donation.

In the present study replacement donors, constituting 91.6% and only 8.4%

were voluntary donors. This is comparable to study done by Kakkar et al (94.7%) 4,

Srikrisnaet al ( 98.5%)1 and Singh et al (84.5%)68.

In contrast predominance of voluntary donors was noted by Bhattacharya et al

(94.6%)69 and Pallavi et al (64.78%)70.

91
 It is shown that replacement donors constitute the largest group of blood

donors in India reflecting lack of awareness among the general population,the

presence of misconceptions and fears associated with donating blood, the lack of

health education and the indifference attitude of the health sector.

Percentage of voluntary and replacement donors in different studies.

Authors Voluntary donors (%) Replacement donors (%)

Srikrishna et al1 1.5% 98.5%

Kakkar et al4 5.3% 94.7%

Singh et al68 15.5% 84.5%

Bhattachary et al69 94.6% 5.4%

Pallavi et al70 64.78% 35.22%

Present study 8.4% 91.6%

SEROPREVALENCE OF HIV

The sexual contact is a major mode of HIV transmission, however blood

donation is also important mode of infection.71

Globally HIV is one of the biggest challenges faced by the health services.

World wide the estimated adult prevalence of HIV is around 0.8% in general

population.72

In India, according to the latest estimates the National adult HIV prevalence is

0.34% in general population and in blood donors 0.28%38 ( NACO,2009)

In the various Indian studies, the seroprevalence of HIV among blood donors

varies from 0.16% to 0.8%.

92
 In our study the seroprevalence for HIV was 0.17% in total donors. The

seroprevalence in a replacement donors was 0.18%. No voluntary donors were

positive fo r HIV. However the difference in seroprevalence among voluntary and

replacement donors was statistically not significant.

Comparision of HIV seroprevalence among donors in different studies

Authors (yrs) Voluntary Replacement Total

Srikrishna et al (1999)1 00% 0.44% 0.44%

Garg et al (2001)71 0.28% 0.46% 0.44%

Kakkar et al (2004)4 00% 0.2% 0.2%

Singha et al (2004)73 0.8% 0.8% 0.8%

Singh et al (2005)74 0.33% 0.64% 0.54%

Matee et al (2006)75 2.0 4.5% 3.8%

Bhattacharya et al (2007)69 - - 0.35%

Arora et al (2010)76 00% 0.3 0.3

Farnandes et al (2010)77 - - 0.06

Kaur et al (2010)78 - - 0.6%

Pallavi et al(2011)70 - - 0.44%

Present study (2011) 00% 0.18% 0.17%

The seroprevalence of HIV in our study in total donor was 0.17%, which is

comparable to the study by Kakkar et al 4 (2004). The seroprevalence in replacement

donors was 0.18% which is comparable to the study by Kakkar et al.4

In our study no voluntary donor was found to be positive for HIV, which is

similar to the finding of Srikrishna et al1, Kakkar et al4 and Arora et al76.

93
 The seroprevance of HIV in various Indian studies ranged from 0.06 to 3.8%.

In our study seroprevalence of HIV is slightly less compared to national data (0.28%).

This can be attributed to strict donors selection criteria

In our study all the seropositive donors were males, which is similar to

the study by Srikrishna et al and Kakkar et al.4

Since no voluntary donors blood units show seropositivity to HIV in our

study, suggest the need for implementing programmes to achieve 100% voluntary

donations.

In India presently WHO strategy 1 is followed for screening blood donors for

HIV. According to this strategy, if the test is negative for HIV antibodies, the blood

unit is considered free of HIV and if reactive the unit is discarded.the donors found

reactive for by initial assay are directed by blood transfusion services to linked

voluntary counseling and Testing centre (VCTC) for counseling and further

confirmatory testing without repeating test in blood bank.

In our present stud y we followed the same strategy. The seroreactive donors

were given the post test counseling and were advised to modify high risk behavior and

to self exclude from future donations. They referred to VCTC for counseling and

further confirmatory testing.

SEROPREVALENCE OF HBV

Hepatitis B virus is the most important causative agent of transfusion

associated hepatitis. India has been placed in the intermediate zone for prevalence of

hepatitis B by WHO (2 -7%)

94
 In previous Indian studies by Srikrishna et al (1999) 1, Sonawane et al (2003)79

and Singh et al (2004)73 observed the seroprevalence of HBsAg among the blood

donors was 1.86%, 4.07% and 1.8% respectively. They concluded that voluntary

donors are comparatively safe donors.

Bhattacharya et al (2007)69 observed a statistically significant increase in

seroprevalence of HBsAg over a of 2 years among the blood donors in Kolkata

(Eastern India ). The observed seroprevalence of HBsAg in 2005 was 1.66%.

In the present study out of the total 16,872 screened blood units 267 were

seropositive for HBsAg with 07 being voluntary donors and 260 being replacement

donors, giving the seroprevalence of 0.49% and 1.58% among voluntary and

replacement donors respectively. However the difference in seroprevalence among

voluntary and replacement donors was statistically not significant.

Comparision of HBsAg seroprevalence among donors in different studies

Authors (yrs) Voluntary Replacement Total
Garg et al (1998)71 2.57% 3.52% 3.44%

Srikrishna et al1 (1999) - - 1.86%

Sonawane et al (2003)79 2.78% 4.84% 4.07%

Sharma et al (2004)80 0.9% 1.26% 1.08%

Singh et al (2004)73 1.4% 1.9% 1.8%

Gupta et al (2006)67 0.77% 2.35% 1.86%

Bhattacharya et al (2007)69 - - 1.66%

Singh et al (2009)68 0.42% 0.65% 0.62%

Arora et al (2010)76 - - 1.70%

Farnandez et al (2010)77 - - 0.34%

Pallavi et al (2010)70 - - 1.27%

Present study 0.49% 1.68% 1.58%

95
 The overall seroprevalence HBsAg in our study (1.58%) correlated well with

those of Bhattacharya et al (2007) and Arora et al (2010) 67. while seroprevalence in

voluntary donors was 0.49%, is similar to those of Singh et al (2009) 68. The

seroprevalance among replacement donors (1.68%) in our study correlates with that of

Singh et al (1.9%)73 and Sharma et al (1.26%)80

The difference in the seropreva lence of HBsAg among voluntary and

replacement donors in the present study suggests, the need for the concrete and non

remunerated voluntary blood donors base in India.

The seropositive HBsAg donors were given post test counseling and enquired

about the past H/o of jaundice they were advised to undergo liver function tests and

serology marker for HBeAg to know the status of their infectivity. They have been

also advised about screening of their family members for HBsAg and immunization.

India is still in the intermediate prevalence zone for HBsAg (2-7%) and

estimated to be a home to over 40 million HBsAg carriers, despite the fact that a safe

and effective vaccine was available since 1982. This is because hepatitis B

vaccination was not a part of our national immunization programme till recently.

SEROPREVALENCE OF HCV

As the screening for HCV has been made mandatory since June 2001,

information on HCV infection among blood donors is sketchy and only few studies

available.

Prevalence of post-transfusion hepatitis in india is around 1%.74

In HCV infection 75-80% reported to progress to chronic hepatitis of which

10- 20% may progress to cirrhosis and hepatocellular carcinoma.38

96
 World wide it is estimated that 3% of the population have been infected with

HCV which means there are 170 million chronic carriers the prevalence of HCV

antibodies in blood donors in developed countries ranges from 0.4% to 2%73

The wide variations of HCV seroprevalence in different studies in India might

due to the use of different generation of ELISA test kits, having different sensitivities

and specificities70. The prevalence estimated to be 1.5% in India.11

Srikrishna et al (1999)1 and Singh et al (2004)73 observed the seroprevalence

of HCV as 1.02% and 0.5% respectively among the blood donors.

Bhattacharya et al (2007)69 noted a statistically significant increase in

seroprevalence of HCV among blood donors over a period of 2 years. The observed

seroprevalence in 2005 was 0.35%.

Singh et al (2004)73 and Matee et al (2006)75 noted that the seroprevalence of

HCV in voluntary donors was less than that in replacement donors, suggesting that the

voluntary donors are safe donors.

In the present study of the total 16,872 screened blood units, 15 units were

seropositive for HCV. All the 15 seropositive blood units were from replacement

donors. None of the screened blood units from voluntary donors were seropositive for

HCV. The seroprevalence HCV among total donors in the present study was 0.09%.

The seroprevalence among replacement donors was 0.10%, while among the

voluntary blood donors the seroprevalence is zero. However the difference in

seroprevalence among voluntary and replacement donors was statistically not

significant.

97
Comparision of HCV seroprevalence among donors in different studies

Authors(yrs) Voluntary Replacement Total

Srikrisna et al (1999)1 - - 1.02

Despande et al - - 0.34%

Kaur et al (2001)4 - - 0.7%

Singh et al (2004)73 0.2% 0.6% 0.5%

Gupta et al (2006)67 0.20 0.65 0.43%

Bhattacharya et al (2007)69 - - 0.35%

Bagga et al (2007)81 0.58% 0.95% 0.88%

Arora et al (2010)76 - - 1.0

Farnandes et al (2010)77 - - 0.06%

Pallavi et al (2011)70 - - 0.23%

Present study 00 0.10% 0.09%

The observed seroprevalence of HCV of 0.09% in the present study is

comparable with that observed by Farnandes et al77 (2010).

Since all the seropositive blood units were from replacement donors and none

of screened blood units from voluntary donors showed seropositivity, the study

suggests the need for collection of blood from voluntary donors.

The prevalence of HCV in preset study is comparatively low . This can be

attributed to implementation of strict donor selection criteria followed by rational use

of blood.

98
SEROPREVALENCE OF SYPHILIS

In India, constant decline in the prevalence of syphilis is observed. In a study

at Chandigarh major decline in syphilis has been observed. The prevalence of syphilis

decreasing from 10.4% in 1977-85 to 2.5% in 1995-96.49

Srikrishna et al (1999)1, Sonawane et al (2003)79 and Singh et al (2005)74 in

their stud ies noted the seroprevalence of syphilis among the blood donors as 1.6%,

0.87% and 0.26% respectively.

Sharma et al (2004)73 in a review article described in detail the changing

pattern of sexually transmitted infection in India. They quote that Choudhari et al

(1995) observed the seroprevalance of syphilis as zero percent among the blood

donors from Lucknow.

Singh et al (2005)74 and Matee et al (2006)75 observed a statistically

significant difference among voluntary and replacement donors suggesting that

voluntary donors are safe donors

In the present study out of the total 16,872 screened blood donors only two

donors blood units showed sero reactivity for syphilis giving the seroprevalence of

0.01%. The seroprevalence in a replacement donors was 0.02%. No voluntary donors

found positive for syphilis. However the difference was statistically not significant.

Comparision of Syphilis seroprevalence among donors in different studies

Author (yrs) Voluntary Replacement Total

Srikrisna et al (1999)1 - - 1.6%

Garg et al(1998)71 0.129% 0.239% 0.22%

Sonawane et al79 (2003) 0.33% 1.12% 0.87%

99
Singh et al (2005)74 1.4% 2.8% 2.6%

Bhattacharya (2007)69 - - 0.8%

Arora et al (2010)76 - - 0.9%

Pallavi et al (2011)70 - - 0.28%

Choudhary et al49 (1995) 00% 00% 00%

Present study 00% 0.02% 0.01%

The seroprevalence of syphilis in our study was 0.01%, which is low

compared to other studies except the study by Choudhary et al 49 which showed, no

donors were positive for syphilis.

This finding of our present study might be due to the declining trends in the

prevalence of syphilis in general population due to improved access to health care,

improved diagnostic means, effacious treatment modalities and general health

awareness among the population especially due to the emergence of HIV.

In our study we have tested the blood units with RPR test. The cardiolipin

antigen antigens used in RPR test may tend to give biological false positive reaction

(BFR) in the conditions like malaria, mumps, measles, lepromatous leprosy, collagen

disease, rheumatoid arthritis, infectious mononucleosis, rubella, leptospirosis,

relapsing fever etc. The seroreactive donors for RPR test were counseled and advised

confirmatory testing by other treponemal test.

PREVALENCE OF MALARIA

Though globally malaria constitutes a big health problem in general

population, the prevalence of malaria among the blood donors is low and ranges from

0% to 0.05%

100
 In a study by Srikrina et al (1999)1, out of the total 8,617 screened blood units

none of the units were positive for malaria.

Similar finding was noted by Sonawane et al (2003)79 at Ambajogai.

Ghouzzi et al (2008)82 studied the result of new ELISA malaria screening. The

observed malaria prevalence was 0.05%

None of the donors in our study were positive for malaria same as that

observed by Srikrishna et al (1999)1 and Sonawone et al (2003)79, Farnandez et al

(2010) 77and Pallavi et al (2011)70

This observed zero percent malaria positivity rate may be the result of proper

scruitiny of prospective donors as well as the use of less sensitive technique for

screening that is peripheral blood smear which requires presence of = 100 parasites/µl

of blood to be detected by microscopy.

This point to the need for the use of more sensitive technique for screening of

malaria to avoid post transfusion malaria particularly in pregnant women and

immunodeficient patients in whom it is fatal. In endemic area, it is recommended that

chemoprophylaxis shoud be given to all recipients.

Coinfection:

Because of the similar transmission mode and the similar high risk patient

population, coinfection of HIV and hepatitis viruses is becoming common clinical

problem. Chronic HBV and HCV is now a leading cause of morbidity and mortality

for HIV infected patients.

101
 Syphilis has also acquired a new potential for morbity and mortality through

association with increased risk of HIV.

Studies related to this, Srikrishna et al1 and Gupta et al67 have observed

definite correlation between HIV and other infections.

There was no coinfection between HIV and other infections noted in our

study.

102
Conclusion
 CONCLUSION

It has been established that the incidence of transfusion transmissible

infections (TTI) decreased considerably after mandatory testing of blood units for

HIV, HbsAg, HCV, Syphilis, and Malaria.

However, the risk of TTI cannot be eliminated completely even after

mandatory testing of blood units because of risk associated with donations during

window period. With advent of nucleic acid amplification techniques (NAT) western

countries have decreased the risk of TTI to a major extent. This decrease the window

period and hence decrease in incidence of TTI. But the cost effectiveness of the NAT

is poor. Its high cost is of concern especially in economically restricted countries.

Our study showed that the seroprevalence of TTI was more in replacement

donors compared to voluntary donors. However it was statistically not significant.

These results suggest that voluntary blood donors services are needed. There

should be an establishment of nationally coordinated blood transfusion services. All

blood should be tested for TTI with reduction in unnecessary blood transfusion. Thus

ensuring safe blood supply to the recipients.

With the implementation of strict donor selection criteria, use of sensitive

screening tests, and establishment of strict guidelines for blood transfusion, it may be

possible to reduce the incidence of TTI in Indian scenario.

103
Summary
 SUMMARY

 A prospective study to evaluate the seropreva lence of HIV, HBsAg, HCV, and

the prevalence of malaria was undertaken between July 2009 to June 2011.

 A total number of 16872 donors blood units were screened.

 Replacement donors constituted 91.6% and remaining 8.4% were voluntary

donors.

 The donors age ranged between 18-60 years with majority (76.2%) in the range of

18-35 years.

 97.5% donors were males and female donors constituted only 2.5%

 The donors were from different strata of society as evidenced by their

occupational status.

 The seroprevalence of HIV was 0.17% in total donors. All the HIV seropositve

blood units were from replacement donors and from males. The seropositity for

HIV was more in married and urban donors.

 The seroprvalence of HBsAg was 1.58% in total donors. The seroprevalence in

replacement donors (1.68%) was more than voluntary donors (0.49%). Out of the

267 HBsAg positive donors 260 were males. The seropositivity for HBsAg was

more in married donors and urban donors.

 The seroprevalence of HCV was 0.09% in total donors and all the positive donors

were replacement donors and males.

 The seroprevalence of syphilis was 0.01% in total donors and all the positive units

were from replacement donors and were males.

 No donors were found positive for malaria parasites.

 Prevalence of all the TTI were more in the age group of 18-35 years.

 In our study there was no coinfection between HIV and other infections.

104
Bibliography
 BIBLIOGRAPHY

1) Srikrishn A, Sitalaxmi S, Domodhar P. How safe are our safe blood donors.

Indian J Pathol Microbial 1999; 42:411-416.

2) NACO. Standards for blood banks and blood transfusion services 2007;33-34.

3) Afsar I, Gungor S, Sener AG. The prevalence of HBV, HCV, and HIV infections

among blood donors of Izmir, Turkey. Indian J Med Microbiol 2008;26:288-290.

4) Kakkar N, Kaur R, Dhanoa J. Vo luntary donors – need for a second look. Indian J

Pathol Microbiol 2004;47:381-83.

5) Makroo RN. Historical overview of transfusion medicine. Textbook of

Compendium of Transfusion medicine. 2010:p.1-5.

6) Bihl F, Castelli D, Marincola F, Dodd RY. Transfuion trnasmitted infections : J

Trans Med 2007;5:25-26.

7) Park K edt. Epidemiology of communicable diseases. In : Parks textbook of

preventive and social medicine 14th edn. Jabalpur M/s Banarsidas Bhanot 2007:p.

285-296.

8) Suryakant AH (edt). HIV/AIDS chapter 24. In: Community medicine (with recent

advances) in 1st edn, Jaypee Brothers Medical Publishers (P) Ltd.. New Delhi

2009:p.439-454.

9) HIV / AIDS historical time line 2000-2007 online 2007 [cited 2008 July; 14

pages) Available from URL http://fda.gov/oashi/aids/miles20.html.

10) Godbole S, Mehendale S. HIV / AIDS epidemic in India, risk factors, risk

behaviours and strategies for prevention and control : Indian J Med Res

2005;121:356-368.

11) HIV Sentinel surveillance and HIV estimation (online) available from URL

http.//www.naconic.in [cited July 2011].

105
12) Fauci AS, Lane HC. Human immunodeficiency virus (HIV) disease : AIDS and

related disorders. In : Braunwald E, Fauci AS, Kasper DL, Hauser SL, Longo DL,

Jameson JL, editors : Harnson’s principles of internal medicine, Newyork: Mc

Graw Hil 2001:p.1852-1913.

13) Maldarelli F, Diagnosis of HIV. In: Mandell GL, Bennett JE, Dolin R, edt.

Principles and practices of infectious diseases. 6th edn, Philadelphia Elsevier

Churchil livingstone 2005;2:1506-1526.

14) Griffith BP, Cumbell S, Mayo DR. Human Immunodeficiency viruses. In :

Murray PR, Baron EJ, Jorgensen JH, Landry ML, Pfaller MH edt. Manual of

Clinical Microbiology, Chapter 83, 9th edn., Washington DC : ASM press

2007;2:1308-1329.

15) Kumar V, Abbas AK,Fausto N, Aster JC etd. Diseases of the immune system. In :

Robbins and Cotran Pathologic basis of disease, 8th edn., Philadelphia, Saunders.

An Imprint of ELSEVIER Inc 2010:p.235-249.

16) Lix, Wainburg MA. Virology of HIV. In : Amstrong D, Cohen J, edt., Infectious

diseases : London Mosby 1999;p571-574..

17) Puneet B, Mathur NK, Molecular biology of HIV. Indian J STD 1997;18:39-47.

18) Sharma VK. Sexually transmitted diseases and AIDS. New Delhi. Viva Books

Pvt. Ltd., 2003;p.77-86.

19) Ananthnarayan R, Paniker CKJ. Human immunodeficiency virus : AIDS In.

Ananthanarayan and Panikers, Text book of Microbiology, 7th edn., Hyderabad.

Orient Longman Publication 2005;p.582-97.

20) Training module for medical officers on TB/HIV central TB division and NACO,

New Delhi 2005;44-45:

106
21) HIV testing manual – Laboratory diagnosis, Biosafety and quality control.

National AIDS Control Organization, New Delhi.

22) Makroo N : Transfusion transmitted diseases. In : compendium of transfusion

Medicine 2009:p.295-296.

23) Joshi PL, Shankat M, Sengupta D, Mishra SN, Chugh S, Banega UK et al. (edt).

Specialists training and reference module 2000: New Delhi; National AIDS

control organization 2000.

24) Kaur P, Basu S. Transfusion- Transmtted infections : Existing and emerging

pathogens. J Post Grad Med 2005;52:146-151.

25) Narayan S. Microbes and blood transfusion. Indian J Med. Microbiol

2001;19:119-26.

26) Deni SM. Dwyre and Holland PV. Hepatitis viruses. In : Barbaraj JA, Regan FA,

Conteras MC edt., Transfusion Microbiology, Cambridge University Press,

Newyork 2008:p.9-24.

27) Park K (edt). Epidemiology of communicable diseases. In : Park’s Textbook of

preventive and social medicine. 19th edn, Jabalpur : M/s Banarsidas Bhanot

2007:p.161-164.

28) Makroo. Transfusion transmitted diseases. In : Compendium of Transfusion

Medicine, 2009:p.267-282.

29) Kumar V, Abbas AK, Fausto N. Aster J (edt). Liver and Biliary tract. In : Robbins

and Contran Pathologic Basis of disease : 8th edn, Philadelphia Saunders : An

Imprint of ELSEVIER 2010:p.833-851.

30) Ananthnarayan R, Paniker CKJ. Hepatitis viruses. In : Ananth Narayan and

Panikers Textbook of microbiology 7th edn, Hyderabad, Orient Longman

Publication, 2005:p.550-56.

107
31) Dienstag JL, Acute viral hepatitis. In : Faucis et al. edt., Harrison’s Principles of

internal medicine 17th edn, Vol. 2, United States McGraw Hill companies

2008;1932-48.

32) Dodd RY, Hepatitis A, B and non A non B and non C viruses. In : Hillyer CD,

Silberstein LE Ness DM. Anderson KC, Robak JD. Edt., Blood Banking and

Transfusion Medicine. Basic Principles and practice 2nd edn., ELSEVIER,

Churchil Livingstone 2007:p.584-590.

33) Horvat RT, Tectmeir GE. Hepatitis B and D viruses. In : Murray PR edt., Manual

of Clinical Microbiology, 9th edn., Washington DC 2009:p.1641-1653.

34) Lee WM. Hepatitis B virus infection. N Eng J Med 2007;337(4):1733-1743.

35) Cendotti D, Allain J. Transfusion Transmitted hepatitis B virus infection. J

Hepatol 2009;51:798-809.

36) Nayak KC. The status and impact of viral hepatitis. A view of global and Indian

Scene. Anuals Natio nal Acad Med Science India. 1985;21:195-206.

37) Jain H, Chakravarthi A, Verma V, Bhalla P. Seroprevalence of hepatitis viruses in

patients infected with HIV: Indian J Pathol Microbiol 2009;52(1):17-19.

38) Dienstag LJ. Acute viral hepatitis. In: Braunwald E, Fauci AS, Kasper DL, Haner

SL, Longo DL, Janesan JL edition. Harrison’s Principles of internal Medicine.,

17th edn., Newyork MacGraw Hill 2008:p. 1933-1948.

39) Kann M. Gerlich WH. Hepatitis B : In : Mahy BWJ, Collier L edt., Topley &

Wilson Microbiology & Microbial Infections 10th edn, Vol.1, Great Britain :

Arnold 2005:p.1160-1187.

40) Baveja CP. Hepatitis viruses. In : Textbook of Microbiology, 2 nd edn, Kala Amb

Up Gupta Y, Gupta D. 2008:p.487-95.

108
41) Dodd RY. Hepatitis C, Chapter 44, In : Hillyer CD, Silberstein LE, Ness DM,

Anderson KC, Robak JD edt., Blood banking and transfusion medicine : Basic

Principles and practice. 2nd edn, ELSEVIER, Churchil Living Stone. P.592-598.

42) Dwyre MD, Holland PV. Hepatitis viruses. Inc Barbara JA, Regan FAM,

Contreras MC edt., Transfusion Microbiology, Cambridge University Press

2008;p.9-24.

43) Narayan S. : Microbes and blood transfusion. Indian J Med, Microbiol

2001;19(3):119-126.

44) Hepatitis C. Global prevalence: Weekly epidemiological record 2009;28:485-565.

45) Simon SP, Mufimer D. Hepatitis C. Chapter 54 . In : Mahe BWJ, Willer L,

Topley & Wilson’s microbiology, Microbial infection 10th edn., Great Britain

Arnold 2005:p.189-1217.

46) Dodd RY. Infectious disease testing : Basic Principles and practical aspects. In :

Hillyer CD, Silber Stein LE, Ness DM, Anderson KK edit., Blood Banking and

Transfusion medicine. 2nd edn, ELSEVIER; Churchill Livingstone. P.218-219.

47) Mcllough J. Transfusion transmitted diseases. Chapter 15. In: McCullough J.

Transfusion Medicine 2nd edn., ELSEVIER Churchill Livingstone 2005;412-415.

48) Lukehart SA. Syphillis. In : Fauci AS, Braunwald E, Kasper DL, Hauser SL,

Longo DL, Jameson JL, Edt., Harrison’s Principles of Internal Medicine. 17 th

edn., Volume 1, MacGraw Hill 2005:p.1038-1054.

49) Sharma VK, Khanpur S. Changing patterns of sexually transmitted infections in

India.. Natl Med J India, 2004;17:310-19.

50) Koneman CW, Allen SD, Janda MW, Schreckumberger PC, Winn WC,

Spirochaetal infections. In : Colour Atlass and text book of diagnostic

microbiology, 6th edn. New York, Lippincott 2006:1126-34.

109
51) Gupta S, Kumar B. Infectious syphilis. In : Sexually transmitted infections: 1 st

edn., New Delhi, Elsevier : 2005:261-95.

52) Penn CW. Treponema: In: Balows A, Duerden BI, Topley and Wilson’s :

Microbiology and Microbial Infections. 9th edn., Vol.2, Great Britain Arnold

1998:p.1258-72.

53) Norris SJ, Edmondson DG. Factors affecting the multiplication and subculture of

T.pallidum in a tissue culture system : Infection and Immunity 1986;53:534-539.

54) Sharma VK, Khundapur S. Epidemiolo gy of STDs In : STD & AIDS, 1st edn.,

New Delhi : Varshtha V, 2003;18-19.

55) Golden MR et al. Update on syphilis resurgence of an old problem. JAMA

2003;290:1510-14.

56) Young N, McMillan A. Syphilis and the endemic treponematosis. In : McMillan

A, Young H, Ogilvie MM, Scott GR, Clinical practice in STI 1 st edn. London :

Elsevier 2002;395-450.

57) Fitzgerald JJ, Dipesh LA, Blanko DR, Miller JV. Attachment of pallidum to

laminin, collagen IV and collagen I and blockage of attachment by immune Sorbt

IgG. Br J Vener dis1984;60:357-63.

58) Gene M, Ledger WJ. Syphilis in pregnancy. Sex Transf 2005;76:73-79.

59) White NJ, Breman JG, Malaria. In : Fauci AS, Kasper DL, Hanser SL, Longo DL,

Janesan JE. Editors. 17th edn., New York, MacGraw Hill 2008:p.1280-1293.

60) World malaria report 2008 : World Health organization.

61) Sharma AK. Bhasin S, Chaturvedis. Predictors of knowledge about malaria in

India. J Vector Borne Disease 2007;44:189-97.

62) Bihl F, Custelli D, Marincola F, Dodd RY, and Brander C. Transfusion

transmitted infections : J Trans Med 2007;5:25.

110
63) Hommel M, Gilles MN, Maria. Malaria. In : Cox FEG, Wakelin D, Gillepse SH,

Despommier DD, Topley & Wilson’s. Microbiology and Microbial Infections.

Parasitology : 10th edn, 2005 : Arolod.pp. 465-525.

64) McAdam AJ, Sharpe AH. Infectious disease. In : Kumar V, Abbas AK. Fausto N,

Aster JC editors : Robbins and cotran : Pathologic basis of disease. 8 th edn. 2010.

Saunders Elsevier : 386-388.

65) Singh G, Seghal R. Transfusion-transmitted parasitic infections : Asian J Transf

Sci 2010;4(2):pp.73-77.

66) Moiz B. Prevention of transfusion Transmitted Malaria in an Endemic area – A

challenge for Blood Banks : Infectious disease Journal of Pakistan 2004;p.96-98.

67) Gupta PK, Kumar H, Basannar DR, Jayaprakash M. Transfusion transmitted

infections in armed forces : Prevalence and trends, MJAFI 2006;62:348-350.

68) Singh K, Bhat S, Shastry S. Trend in seroprevalence of hepatitis B virus infection

among blood donors of coastal Karanataka, Indian J Infect Dev Ctries

2009;3:376- 379.

69) Bhattacharya P, Chandra PK, Datta S, Banarjee A et al. Significant increase in

HBV, HCV, HIV and syphilis infection among blood donors in West Bengal,

Eastern India, 2004-2005. Exploratory screening reveals high frequency of occult

HBV infection. World J Gastroenterol 13:3730-3733.

70) Pallavi P, Ganesh CK, Jayashree K, Manjunath GV. Seroprevalence and trends in

transfusion transmitted infections among blood donors in a University hospital

Blood Bank : A 5 year study. Indian J Hematol, Blood Transfus 2011;27(1):1-6.

71) Garg S, Mathur DR, Garg DK. Comparision of seropositivity of HIV, HBV, HCV,

and syphilis in replacement and voluntary blood donors in Western India. Indian J

Pathol / Microbiol 2001;44(4):409-412.

111
72) HIV/AIDS estimates are revised downwards; world health statistics 2008:p.13-14.

73) Singh B, Katriya S, Gupta R. Infectious markers in blood donors of East Delhi :

Prevalence and Trends. Indian J Pathol / Microbiol 2004;47(4):477-479.

74) Singh B, Verma M, Kotru M, Verma K, Batra M. Prevalence of HIV and VDRL

seropositivity in blood donors of Delhi. Indian J Med Res 2005;122:234-236.

75) Matee M, Magesa P, Lyamuya E. Seroprevalence of human immunodeficiency

virus, hepatitis B and C viruses, and syphilis infectio ns among blood donors at the

Muhimbili National Hospital in Dar Es Salaam, Tanzania BMC Public Health

2006;6:21.

76) Arora D, Arora B, Kheterpal A. Seroprevalence of HIV, HBV, HCV and syphilis

in blood donors in Southern Haryana. Indian J Pathol Microbiol 2010;53(2):308-

309.

77) Farnandez H, D’Souza PF, D’Souza PM. Prevalence of transfusion transmitted

infections in voluntary and replacement donors. Indian J Hematol, Blood

Transfusion 2010;26(3):89-91.

78) Kaur G, Basu S, Kumar R, Kumar P, Garg S. Patterns of infections among blood

donors in tertiary care centre : A retrospective study. The Natl Med J India,

2010;23(3):147-149.

79) Sonawane BR, Birare SD, Kulkarni PV. Prevalence of seroreactivity among blood

donors in rural population. Indian J Med Sci 2003;57:405-407.

80) Sharma R, Cheema R, Vajpayee M, Rao U, Kumar S et al. Prevalence of markers

of transfusion transmissible diseases in voluntary and replacement donors. The

Natl med J of India 2004;17:19-21.

112
81) Bagga PK, Singh SP et al. Seroprevalence of hepatitis C antibodies in healthy

blood donors – a prospective study. Indian J Pathol Microbiol 2007;50(2):429-

432.

82) Gouzzi MH. Beolet M, Banet V et al. Results of new ELISA malaria, screening in

French blood bank. Impact on deferral rate in 2006-07; Von Sang 2008;95:307.

113
Annexures
 PROFORMA
Name : Age: Sex:
Address : Occupation :
Marital status : Single/Married

1. CPD Bag No :
2. Date of collection :
3. Type of donor : Voluntary Replacement

MEDICAL HISTORY :
1. H/o previous donation & Interval : Yes / No

2. Recent h/o fever/common cold : Yes / No

3. H/o malarial fever in last 3 months : Yes / No

4. H/o jaundice, infective hepatitis in last 2 years : Yes / No

5. H/o DM/HT/Tuberculosis : Yes / No

6. H/o Convulsions/ Recent drug intake : Yes / No

7. H/o significant weight loss in last 6 months : Yes / No

8. H/o vaccination in last 6 months : Yes /

No Nature of vaccination

9. H/o received blood transfusion or its components : Yes / No

10. H/o any major surgery in last 6 months : Yes / No

11. H/o minor surgery in last 3 months : Yes / No

12. Date of last menstrual period :

13. Date of pregnancy/ breast feeding / abortion :

114
GENERAL EXAMINATION :
1. Weight kgs
2. Height cms
3. Pulse rate /min
4. Temperature °C
5. Blood pressure mm of Hg
6. Venepuncture site

SYSTEMIC EXAMINATION :
1. Central nervous system

2. Cardiovascular system

3. Respiratory sys tem

4. Per abdomen

LABORATORY TESTS :
1. Hemoglobin gm%
2. ABO and Rh blood grouping
3. CPD bag No. and Date of collection
4. Screening tests

HIV HBsAg HCV Syphilis (RPR) MP

Positive Positive Positive Reactive Posit ive
Negative Negative Negative Non reactive Negative

115
 MASTER CHART
VD/ Serological tests
Sl. No B.B.No Age Sex U/R Occupation S/M Bl GR MP
RP HIV HBsAg HCV RPR
1 3677/09 28 M U Labour Ma RP B Positive Negative Positive Negative Negative Negative
2 3754/09 34 M R student Ma RP O Positive Negative Positive Negative Negative Negative
3 3788/09 33 M R Business Ma RP AB Positive Negative Positive Negative Negative Negative
4 3825/09 26 M R Carpenter S RP B Positive Negative Positive Negative Negative Negative
5 3875/09 30 M R Business Ma RP O Positive Negative Positive Negative Negative Negative
6 4023/09 23 M U PC S RP B Positive Negative Positive Negative Negative Negative
7 4187/09 23 M R Agriculture S RP O Positive Negative Positive Negative Negative Negative
8 4221/09 34 M R Agriculture Ma RP B Positive Negative Positive Negative Negative Negative
9 4225/09 21 M R Cook S RP A Positive Positive Negative Negative Negative Negative
10 4343/09 28 M R Barber Ma RP O Positive Negative Positive Negative Negative Negative
11 4409/09 23 M R Driver Ma RP AB Positive Negative Positive Negative Negative Negative
12 4429/09 20 M R Coolie Ma RP O Positive Negative Positive Negative Negative Negative
13 4469/09 25 M U Driver Ma RP B Positive Negative Negative Positive Negative Negative
14 4476/09 35 M U Driver Ma RP O Positive Negative Positive Negative Negative Negative
15 4682/09 36 M R Agriculture Ma RP O Positive Negative Positive Negative Negative Negative
16 4736/09 32 M R Coolie Ma VD O Positive Negative Positive Negative Negative Negative
17 4739/09 27 M U Prof Ma VD A Negative Negative Positive Negative Negative Negative
18 4741/09 33 M R Prof Ma VD A Positive Negative Positive Negative Negative Negative
19 4767/09 34 M U Business Ma RP O Positive Negative Positive Negative Negative Negative
20 4804/09 28 M U Prof S RP B Positive Negative Positive Negative Negative Negative
21 4812/09 24 M R student S RP B Positive Negative Positive Negative Negative Negative
22 4845/09 28 M R Business Ma RP B Positive Negative Positive Negative Negative Negative
23 4862/09 23 M U student S RP B Positive Negative Positive Negative Negative Negative
24 4904/09 21 M U Driver S RP B Positive Negative Positive Negative Negative Negative
25 4924/04 19 M U student S RP O Positive Negative Negative Positive Negative Negative
26 4981/09 38 M U Driver Ma RP B Positive Negative Negative Positive Negative Negative
27 5009/09 32 M U Coolie Ma RP O Positive Negative Positive Negative Negative Negative
28 5038/09 34 M U Teacher Ma RP AB Positive Negative Positive Negative Negative Negative
29 5151/09 34 M U Prof Ma RP A Positive Negative Positive Negative Negative Negative
30 5180/09 34 M R Coolie Ma RP A Positive Positive Negative Negative Negative Negative
31 5187/09 19 M U student S RP O Positive Negative Positive Negative Negative Negative
32 5308/09 23 M U student S RP O Positive Negative Positive Negative Negative Negative
33 5329/09 36 M R Business Ma RP B Positive Negative Positive Negative Negative Negative
34 5337/09 25 M U Business Ma RP O Positive Negative Positive Negative Negative Negative
35 5344/09 24 M R Coolie S RP B Positive Negative Positive Negative Negative Negative
36 5357/09 37 M U Business Ma RP B Positive Negative Positive Negative Negative Negative
37 5368/09 32 M U Teacher Ma RP O Negative Negative Positive Negative Negative Negative
38 5414/09 27 M U Teacher S RP B Positive Negative Positive Negative Negative Negative
39 5524/09 42 M R Agriculture Ma RP O Positive Negative Positive Negative Negative Negative
40 5534/09 23 M R Coolie Ma RP B Positive Negative Positive Negative Negative Negative
41 5574/09 22 M U Driver S RP AB Positive Negative Positive Negative Negative Negative
42 5578/09 25 M R Agriculture Ma RP O Positive Negative Positive Negative Negative Negative
43 5642/09 25 M R Agriculture Ma RP B Positive Negative Positive Negative Negative Negative
44 5802/09 29 M R Agriculture Ma RP B Positive Positive Negative Negative Negative Negative
45 5904/09 28 M U Buissness Ma RP B Positive Negative Positive Negative Negative Negative
46 5926/09 22 M U Photo shop Ma RP O Positive Negative Positive Negative Negative Negative
47 5931/09 20 M U Workshop Ma RP O Positive Negative Positive Negative Negative Negative
48 6032/09 24 M R Coolie S RP O Positive Negative Positive Negative Negative Negative
49 6035/11 26 M R Driver Ma RP B Positive Negative Positive Negative Negative Negative
50 6089/09 23 M U student S RP A Positive Negative Positive Negative Negative Negative
51 6099/09 32 M R Coolie Ma RP O Positive Negative Positive Negative Negative Negative
52 6175/09 26 M R Agriculture S RP A Positive Negative Positive Negative Negative Negative
53 6193/09 41 M R Agriculture Ma RP B Positive Negative Positive Negative Negative Negative
54 6366/09 20 M U student S VD AB Positive Negative Positive Negative Negative Negative
55 6447/09 24 M U Business S RP B Positive Negative Positive Negative Negative Negative
56 6454/09 45 M U Teacher Ma RP O Positive Negative Positive Negative Negative Negative
57 6460/09 34 M U Contracter Ma RP A Positive Negative Positive Negative Negative Negative
58 6487/09 24 M U student S RP AB Positive Negative Positive Negative Negative Negative
59 6488/09 44 M R Agriculture Ma RP A Positive Negative Positive Negative Negative Negative
60 6507/09 26 M U Labour S RP B Positive Positive Negative Negative Negative Negative
61 6534/09 45 M R Labour Ma RP B Positive Negative Negative Positive Negative Negative
62 6635/09 33 M R Business Ma RP A Positive Negative Positive Negative Negative Negative
63 6638/09 26 M U Carpenter Ma RP B Positive Negative Positive Negative Negative Negative
64 6685/09 25 M R Agriculture Ma RP B Positive Negative Positive Negative Negative Negative
65 6694/09 34 M U Teacher Ma RP O Negative Negative Positive Negative Negative Negative
66 6738/09 27 M U Prof Ma RP O Positive Negative Negative Positive Negative Negative
67 6787/09 20 M R student Ma RP O Positive Negative Positive Negative Negative Negative
68 6813/09 40 M R Agriculture Ma RP AB Positive Positive Negative Negative Negative Negative
69 6830/09 32 M R Agriculture Ma RP A Positive Negative Positive Negative Negative Negative
70 6894/09 50 M R Agriculture Ma RP AB Positive Negative Negative Negative Negative Negative
71 6896/09 31 M U Teacher Ma RP B Positive Negative Positive Negative Negative Negative
72 6977/09 30 M U Business Ma RP AB Positive Negative Positive Negative Negative Negative
73 7039/09 40 M U Prof Ma RP A Positive Negative Positive Negative Negative Negative
74 7090/ 09 30 F U Houswife Ma RP A Positive Negative Positive Negative Negative Negative
75 7103/09 26 M R Agriculture Ma RP O Positive Negative Positive Negative Negative Negative
76 7126/09 41 M R Agriculture Ma RP A Negative Negative Negative Positive Negative Negative
77 7187/09 27 M R Agriculture Ma RP O Positive Negative Positive Negative Negative Negative
78 7251/09 49 M R Agriculture Ma RP O Positive Negative Positive Negative Negative Negative
79 7254/09 22 M R watchman S RP A Positive Negative Negative Positive Negative Negative
80 7262/09 22 M U Labour S RP B Positive Negative Positive Negative Negative Negative
81 7320/09 26 M R Agriculture Ma RP A Positive Negative Positive Negative Negative Negative
82 7345/09 24 M R Agriculture S RP O Positive Negative Positive
83 7350/09 24 M U student S RP O Positive Negative Positive Negative Negative Negative
84 7488/09 21 M R student S RP O Positive Negative Positive Negative Negative Negative
85 7494/09 40 M R Agriculture Ma RP B Positive Positive Negative Negative Negative Negative
86 7574/09 26 M R Kiranashop Ma RP B Positive Negative Positive Negative Negative Negative
87 7586/09 34 M R Buissness Ma RP AB Positive Negative Positive Negative Negative Negative
88 102/10 23 M U Student S RP B Positive Negative Positive Negative Negative Negative
89 218/10 28 M U Driver Ma RP A Positive Negative Positive Negative Negative Negative
90 235/10 22 M R student S RP A Positive Negative Positive Negative Negative Negative

116
 91 362/10 24 M R Agriculture Ma RP O Positive Negative Positive Negative Negative Negative
92 366/10 23 M R Painter Ma RP A Positive Negative Positive Negative Negative Negative
93 369/10 32 M U Driver Ma RP B Positive Negative Positive Negative Negative Negative
94 467/10 29 M R Farmer Ma RP B Positive Negative Positive Negative Negative Negative
95 491/10 25 M U Shop S RP A Positive Negative Positive Negative Negative Negative
96 548/10 25 M U watchman Ma RP O Positive Negative Positive Negative Negative Negative
97 577/10 37 M R Agriculture Ma RP O Positive Negative Positive Negative Negative Negative
98 706/10 32 M R SDC Ma RP B Positive Negative Positive Negative Negative Negative
99 823/10 22 M U Driver S RP O Positive Positive Negative Negative Negative Negative
100 836/10 30 F U Tailor Ma RP B Positive Negative Positive Negative Negative Negative
101 843/10 29 M R Farmer Ma RP A Positive Negative Positive Negative Negative Negative
102 845/10 35 M R Business Ma RP O Positive Negative Negative Positive Negative Negative
103 906/10 48 M R Agriculture Ma RP B Positive Negative Positive Negative Negative Negative
104 933/10 23 M R Business Ma RP B Positive Negative Positive Negative Negative Negative
105 1196/10 50 M R Agriculture Ma RP O Negative Positive Negative Negative Negative Negative
106 1245/10 32 M R Tailor Ma RP B Positive Negative Positive Negative Negative Negative
107 1303/10 33 M R Buissness Ma RP A Positive Negative Negative Positive Negative Negative
108 1357/10 27 M R Agriculture Ma RP O Positive Negative Positive Negative Negative Negative
109 1358/10 28 M R Agriculture Ma RP A Positive Negative Positive Negative Negative Negative
110 1436/10 30 M R Business Ma RP A Positive Negative Positive Negative Negative Negative
111 1637/10 26 M U Driver S RP A Positive Negative Positive Negative Negative Negative
112 1651/10 32 M R Business Ma RP O Positive Negative Positive Negative Negative Negative
113 1677/10 32 M U Business Ma RP A Positive Negative Positive Negative Negative Negative
114 1768/10 23 M U Tailor S RP AB Positive Negative Positive Negative Negative Negative
115 1826/10 45 M U Coolie Ma RP O Positive Negative Positive Negative Negative Negative
116 1892/10 34 M U Agriculture Ma RP B Positive Negative Positive Negative Negative Negative
117 1934/10 38 M U Buissness Ma RP A Positive Negative Positive Negative Negative Negative
118 2073/10 27 M R Driver Ma RP O Positive Negative Positive Negative Negative Negative
119 2075/10 20 M R student S RP B Positive Negative Positive Negative Negative Negative
120 2102/10 28 M U Driver Ma RP A Positive Positive Negative Negative Negative Negative
121 2178/10 34 M R Agriculture Ma RP O Positive Negative Positive Negative Negative Negative
122 2195/10 35 M U Kiranashop Ma RP AB Positive Negative Positive Negative Negative Negative
123 2291/10 32 M U Coolie Ma RP B Positive Negative Positive Negative Negative Negative
124 2322/10 32 M U Business Ma RP O Positive Negative Positive Negative Negative Negative
125 2336/10 29 M R Farmer Ma RP A Positive Negative Positive Negative Negative Negative
126 2361/10 23 M U student S RP O Positive Negative Positive Negative Negative Negative
127 2385/10 30 M U Business Ma RP A Positive Negative Positive Negative Negative Negative
128 2417/10 47 M R Tailor Ma RP A Positive Negative Positive Negative Negative Negative
129 2578/10 22 M R student S RP O Positive Negative Positive Negative Negative Negative
130 2602/10 23 M R student S RP O Positive Negative Positive Negative Negative Negative
131 2605/10 35 M R Agriculture Ma RP A Positive Negative Positive Negative Negative Negative
132 2785/10 19 M U student S RP A Positive Negative Positive Negative Negative Negative
133 3000/10 23 M U student S RP A Positive Negative Positive Negative Negative Negative
134 3012/10 35 M R Agriculture Ma RP O Positive Negative Positive Negative Negative Negative
135 3022/10 29 M U Painter Ma RP A Negative Negative Positive Negative Negative Negative
136 3078/10 35 M R Teacher Ma RP A Positive Negative Positive Negative Negative Negative
137 3168/10 32 M R Farmer Ma RP B Positive Negative Positive Negative Negative Negative
138 3171/10 26 M U Driver S RP O Positive Positive Negative Negative Negative Negative
139 3359/10 35 M U Prof Ma RP B Positive Negative Positive Negative Negative Negative
140 3364/10 46 M R Agriculture Ma RP O Positive Negative Positive Negative Negative Negative
141 3390/10 38 M U Business Ma RP B Positive Negative Positive Negative Negative Negative
142 3555/10 32 M U Driver Ma RP A Positive Positive Negative Negative Negative Negative
143 3633/10 24 M R Shop S RP O Positive Negative Positive Negative Negative Negative
144 3668/10 33 M R Labour Ma RP O Negative Negative Positive Negative Negative Negative
145 3684/10 21 M R student S RP B Positive Negative Positive Negative Negative Negative
146 3793/10 21 M R student S RP B Positive Negative Positive Negative Negative Negative
147 4026/10 39 M R Coolie Ma RP A Positive Negative Positive Negative Negative Negative
148 4162/10 38 M R Farmer Ma RP B Positive Negative Positive Negative Negative Negative
149 4189/10 40 M R Farmer Ma RP B Positive Negative Positive Negative Negative Negative
150 4226/10 23 M U student S RP A Positive Negative Positive Negative Negative Negative
151 4247/10 22 M U Student S RP A Positive Negative Positive Negative Negative Negative
152 4287/10 25 M R Coolie Ma RP A Positive Negative Positive Negative Negative Negative
153 4480/10 33 M R Farmer Ma RP B Negative Negative Positive Negative Negative Negative
154 4526/10 25 M U Business S RP B Positive Negative Positive Negative Negative Negative
155 4546/10 20 M R student S RP O Positive Negative Positive Negative Negative Negative
156 4598/10 22 M R student S RP O Positive Negative Positive Negative Negative Negative
157 4608/10 20 M U student Ma RP O Positive Negative Positive Negative Negative Negative
158 4643/10 28 M R Agriculture Ma RP B Positive Negative Positive Negative Negative Negative
159 4735/10 41 M R Agriculture Ma RP O Positive Negative Positive Negative Negative Negative
160 4790/10 37 M R Driver Ma RP A Positive Positive Negative Negative Negative Negative
161 4949/10 30 M R Farmer Ma RP B Positive Negative Positive Negative Negative Negative
162 5047/10 26 M R Farmer Ma RP A Positive Negative Positive Negative Negative Negative
163 5091/10 31 M U Business Ma RP B Positive Positive Negative Negative Negative Negative
164 5092/10 30 M U Prof Ma RP B Positive Negative Positive Negative Negative Negative
165 5098/10 25 M U student S RP B Positive Negative Positive Negative Negative Negative

117
166 5099/10 34 M R Coolie Ma RP B Positive Negative Positive Negative Negative Negative
167 5160/10 34 M U Teacher Ma RP A Positive Negative Positive Negative Negative Negative
168 5178/10 25 M U student S RP A Positive Negative Positive Negative Negative Negative
169 5191/10 50 M R Agriculture Ma RP B Positive Negative Positive Negative Negative Negative
170 5278/10 60 M U Prof Ma RP B Positive Negative Positive Negative Negative Negative
171 5392/10 29 M R Farmer Ma RP A Positive Negative Positive Negative Negative Negative
172 5426/10 40 M U Business Ma RP B Positive Negative Positive Negative Negative Negative
173 5453/10 22 M U student Ma RP O Positive Negative Positive Negative Negative Negative
174 5483/10 30 M U Prof Ma RP O Positive Negative Positive Negative Negative Negative
175 5484/10 35 M R Agriculture Ma RP A Positive Positive Negative Negative Negative Negative
176 5536/10 40 M R Coolie Ma RP AB Positive Negative Positive Negative Negative Negative
177 5620/10 32 M U Business Ma RP O Positive Negative Positive Negative Negative Negative
178 5709/10 34 M R Agriculture Ma RP B Positive Negative Positive Negative Negative Negative
179 5738/10 27 M R Teacher S RP AB Positive Negative Positive Negative Negative Negative
180 5756/10 23 M U Driver S RP A Positive Positive Negative Negative Negative Negative
181 5781/10 30 M U Kiranashop Ma RP B Positive Negative Positive Negative Negative Negative
182 5796/10 19 M U student Ma RP B Positive Negative Positive Negative Negative Negative
183 5805/10 21 M R student S RP AB Positive Negative Positive Negative Negative Negative
184 5982/10 32 M U Teacher Ma RP A Positive Negative Positive Negative Negative Negative
185 6290/10 34 M U Business Ma RP O Positive Negative Positive Negative Negative Negative
186 6352/10 34 M R Agriculture Ma RP A Positive Negative Positive Negative Negative Negative
187 6360/10 35 M R Agriculture Ma RP A Positive Negative Positive Negative Negative Negative
188 6433/10 27 M U Coolie Ma RP B Positive Negative Positive Negative Negative Negative
189 6456/10 37 M R Supervisor Ma RP B Positive Negative Positive Negative Negative Negative
190 6483/10 28 M U Painter Ma RP A Positive Negative Positive Negative Negative Negative
191 6509/10 30 M R Farmer Ma RP A Positive Negative Positive Negative Negative Negative
192 6571/10 23 M U Business S RP O Positive Negative Positive Negative Negative Negative
193 6736/10 48 M R Mutton shop Ma RP A Positive Positive Negative Negative Negative Negative
194 6938/10 27 M R com work S RP B Positive Positive Negative Negative Negative Negative
195 6953/10 40 M R Farmer Ma RP O Positive Negative Positive Negative Negative Negative
196 6975/10 23 M R Driver S RP B Positive Negative Positive Negative Negative Negative
197 6980/10 26 M R Electrician S RP O Positive Positive Negative Negative Negative Negative
198 7018/10 35 M U Workshop Ma RP A Positive Negative Positive Negative Negative Negative
199 7025/10 50 M R Teacher Ma RP O Positive Negative Negative Positive Negative Negative
200 7033/10 42 M R Teacher Ma RP O Positive Negative Positive Negative Negative Negative
201 7083/10 27 M R Tailor Ma RP AB Positive Negative Negative Positive Negative Negative
202 7120/10 29 M U Business Ma RP O Positive Negative Positive Negative Negative Negative
203 7163/10 37 M U Business Ma RP B Positive Negative Positive Negative Negative Negative
204 7218/10 26 M R Kiranashop Ma RP O Positive Negative Positive Negative Negative Negative
205 7286/10 32 M U Kiranashop Ma RP O Positive Negative Positive Negative Negative Negative
206 7316/10 32 M R Agriculture Ma RP A Positive Negative Positive Negative Negative Negative
207 7327/10 32 M R Agriculture Ma RP O Positive Negative Positive Negative Negative Negative
208 7402/10 25 M U Labour S RP B Positive Positive Negative Negative Negative Negative
209 7435/10 34 M R Agriculture Ma RP B Positive Negative Positive Negative Negative Negative
210 7478/10 29 M U Prof Ma RP O Positive Negative Positive Negative Negative Negative
211 7501/10 28 M R Farmer Ma RP A Positive Positive Negative Negative Negative Negative
212 7547/10 29 M U Mechanic Ma RP O Positive Negative Positive Negative Negative Negative
213 7698/10 36 M U Business Ma RP A Positive Negative Positive Negative Negative Negative
214 7856/10 42 M U Business Ma RP B Positive Negative Positive Negative Negative Negative
215 7857/10 33 M R Agriculture Ma RP O Negative Negative Positive Negative Negative Negative
216 7892/10 24 M U student Ma RP O Positive Negative Positive Negative Negative Negative
217 7964/10 42 M R Hotel Ma RP B Positive Negative Positive Negative Negative Negative
218 7969/10 19 M U student S RP O Positive Negative Positive Negative Negative Negative
219 8060/10 20 M U student S VD O Positive Negative Positive Negative Negative Negative
220 8091/10 23 M U student S VD B Positive Negative Positive Negative Negative Negative
221 8208/10 24 M U student S RP B Positive Negative Positive Negative Negative Negative
222 8226/10 24 M R Agriculture S RP A Positive Negative Positive Negative Negative Negative
223 8279/10 32 M U Business Ma RP B Positive Negative Positive Negative Negative Negative
224 8312/10 35 M U Business Ma RP O Positive Negative Negative Positive Negative Negative
225 8403/10 28 M U Labour Ma RP O Positive Positive Negative Negative Negative Negative
226 8527/10 27 M R Agriculture Ma RP A Positive Negative Positive Negative Negative Negative
227 8628/10 19 M U student S RP A Positive Negative Positive Negative Negative Negative
228 8668/10 37 M R Coolie Ma RP AB Positive Positive Negative Negative Negative Negative
229 8699/10 25 M U student S VD O Positive Negative Positive Negative Negative Negative
230 8856/10 26 M U Business Ma RP O Positive Negative Positive Negative Negative Negative
231 8949/10 29 M U Business Ma RP O Positive Negative Positive Negative Negative Negative
232 8998/10 33 M U Engineer Ma RP A Positive Negative Positive Negative Negative Negative
233 9104/10 30 M U Agriculture Ma RP AB Positive Negative Positive Negative Negative Negative
234 9175/10 30 M U Mechanic Ma RP A Positive Negative Positive Negative Negative Negative
235 9177/10 39 M R Agriculture Ma RP B Positive Negative Positive Negative Negative Negative
236 9239/10 26 M R Agriculture Ma RP A Positive Negative Positive Negative Negative Negative
237 9302/10 30 M U Teacher Ma RP B Positive Negative Positive Negative Negative Negative
238 9392/10 31 M R Agriculture Ma RP B Positive Negative Positive Negative Negative Negative
239 012/11 23 M U student S RP A Positive Negative Positive Negative Negative Negative
240 042/11 26 M U Business S RP AB Positive Negative Positive Negative Negative Negative
241 043/11 25 M U Kir anashop S RP B Positive Negative Positive Negative Negative Negative
242 356/11 34 M U Prof Ma RP O Positive Negative Positive Negative Negative Negative
243 389/11 43 M R Agriculture Ma RP O Positive Negative Positive Negative Negative Negative
244 489/11 35 M R Agriculture Ma RP A Positive Negative Positive Negative Negative Negative
245 504/11 20 M U student S RP B Positive Negative Positive Negative Negative Negative
246 537/11 22 M U student S RP B Positive Negative Positive Negative Negative Negative
247 647/11 27 M U Driver Ma RP A Positive Positive Negative Negative Negative Negative
248 703/11 36 M U Driver Ma RP A Positive Positive Negative Negative Negative Negative
249 986/11 28 M U Coolie Ma RP B Positive Negative Positive Negative Negative Negative
250 1044/11 33 M U Business Ma RP O Positive Negative Positive Negative Negative Negative
251 1231/11 27 M R Driver Ma RP O Positive Negative Positive Negative Negative Negative
252 1293/11 35 M U Driver Ma RP A Positive Negative Negative Positive Negative Negative
253 1314/11 19 M U student S RP B Positive Negative Positive Negative Negative Negative
254 1329/11 20 M U student S RP O Positive Negative Negative Positive Negative Negative
255 1340/11 37 M R Agriculture Ma RP B Positive Negative Positive Negative Negative Negative
256 1381/11 27 M R Cook S RP A Positive Positive Negative Negative Negative Negative
257 1390/11 20 M U Coolie S RP A Positive Negative Positive Negative Negative Negative
258 1412/11 28 M R Farmer Ma RP O Positive Negative Positive Negative Negative Negative

118
259 1478/11 45 M U Mutton shop Ma RP O Negative Positive Negative Negative Negative Negative
260 1510/11 26 M U watchman S RP B Positive Positive Negative Negative Negative Negative
261 1542/11 19 M U student S RP O Positive Negative Positive Negative Negative Negative
262 1560/11 23 M U Agriculture S RP O Positive Negative Positive Negative Negative Negative
263 1714/11 22 M U Coolie S RP B Positive Negative Positive Negative Negative Negative
264 1716/11 22 M U Coolie S RP O Positive Negative Positive Negative Negative Negative
265 1767/11 32 M U Agriculture Ma RP O Positive Negative Positive Negative Negative Negative
266 1780/11 29 M U Dry cleaner Ma RP O Positive Negative Positive Negative Negative Negative
267 1787/11 33 M U Store Ma RP A Positive Negative Positive Negative Negative Negative
268 1885/11 26 M U Business S RP O Positive Negative Positive Negative Negative Negative
269 1941/11 29 M U Teacher Ma RP B Positive Negative Positive Negative Negative Negative
270 1952/11 35 M R Agriculture Ma RP O Positive Negative Positive Negative Negative Negative
271 1954/11 36 M R Agriculture Ma RP B Positive Negative Negative Positive Negative Negative
272 1963/11 20 M U student S RP O Positive Negative Positive Negative Negative Negative
273 1982/11 20 M R student S RP O Positive Negative Positive Negative Negative Negative
274 1987/11 30 M U Farmer Ma RP B Positive Negative Positive Negative Negative Negative
275 1999/11 30 M U Business Ma RP O Positive Negative Positive Negative Negative Negative
276 2039/11 29 M U Teacher Ma RP O Positive Negative Positive Negative Negative Negative
277 2043/11 30 M R Agriculture Ma RP O Positive Negative Positive Negative Negative Negative
278 2064/11 32 M U Business Ma RP O Positive Negative Positive Negative Negative Negative
279 2082/11 23 M U student S RP A Positive Negative Positive Negative Negative Negative
280 2109/11 38 M R Agriculture Ma RP O Positive Negative Positive Negative Negative Negative
281 2147/11 40 M R Agriculture Ma RP O Positive Negative Positive Negative Negative Negative
282 2225/11 21 M R student S RP A Positive Negative Positive Negative Negative Negative
283 2265/11 22 M U Electrician S RP O Positive Negative Positive Negative Negative Negative
284 2466/11 29 M U Business Ma RP B Positive Negative Positive Negative Negative Negative
285 2481/11 53 M U Business Ma RP A Positive Negative Positive Negative Negative Negative
286 2517/11 30 M R Agriculture Ma RP A Positive Negative Positive Negative Negative Negative
287 2521/11 20 M U Driver S RP AB Positive Negative Positive Negative Negative Negative
288 2539/11 30 M R Agriculture Ma RP A Positive Negative Positive Negative Negative Negative
289 2642/11 47 M R Agriculture Ma RP B Positive Negative Positive Negative Negative Negative
290 2643/11 27 M R Agriculture Ma RP B Positive Negative Negative Negative Positive Negative
291 2657/11 29 M R Computer Ma RP A Positive Negative Positive Negative Negative Negative
292 2687/11 22 M U Electrician S RP B Positive Negative Negative Negative Positive Negative
293 2691/11 41 M R Business Ma RP B Positive Negative Positive Negative Negative Negative
294 2707/11 28 M U Business Ma RP AB Positive Negative Positive Negative Negative Negative
295 2716/11 35 F U PRO Ma RP A Positive Negative Positive Negative Negative Negative
296 2857/11 29 M R Driver Ma RP O Positive Negative Positive Negative Negative Negative
297 2957/11 38 M R Agriculture Ma RP A Positive Negative Positive Negative Negative Negative
298 2982/11 29 M U Teacher Ma RP B Positive Negative Positive Negative Negative Negative
299 3001/11 23 M U student S RP AB Positive Negative Positive Negative Negative Negative
300 3016/11 29 M U Agriculture Ma RP O Positive Negative Positive Negative Negative Negative
301 3145/11 38 M U Business Ma RP A Positive Negative Positive Negative Negative Negative
302 3205/11 35 M R Business Ma RP A Positive Negative Positive Negative Negative Negative
303 3210/11 40 M U Teacher Ma RP A Positive Negative Positive Negative Negative Negative
304 3216/11 45 M R Agriculture Ma RP B Positive Negative Positive Negative Negative Negative
305 3232/11 34 M U Business Ma RP B Positive Negative Positive Negative Negative Negative
306 3269/11 45 M R Agriculture Ma RP B Positive Negative Positive Negative Negative Negative
307 3307/11 32 M U watchman Ma RP B Positive Positive Negative Negative Negative Negative
308 3320/11 28 M U Business Ma RP A Positive Negative Positive Negative Negative Negative
309 3332/11 33 M U Business Ma RP O Positive Negative Positive Negative Negative Negative
310 3385/11 35 M U Mechanic Ma RP O Positive Negative Positive Negative Negative Negative
311 3387/11 27 M U Teacher Ma RP O Positive Negative Positive Negative Negative Negative
312 3393/11 21 M R Agriculture S RP O Positive Negative Positive Negative Negative Negative

119
 KEY TO MASTER CHART

U – Urban

R – Rural

M – Male

F – Female

S – Single

Ma – Married

VD – Voluntary donor

RP – Replacement donor

HIV – Human immunodeficiency virus

HBsAg – Hepatitis B surface antigen

HCV – Hepatitis C virus

RPR – Rapid plasma

reagin Mp – Malaria Parasite

PC – Police constable

Prof. – Professional.

120