Comparative Study of Hiv, Hcv and Hbv Screening by Third and Fourth Generation Elisa in Donated Blood

COMPARATIVE STUDY OF HIV, HCV AND HBV SCREENING BY THIRD AND FOURTH GENERATION ELISA IN DONATED BLOOD

Dissertation submitted to

THE TAMILNADU Dr.M.G.R. MEDICAL UNIVERSITY

In partial fulfilment of the regulations

For the award of the degree of

M.D BRANCH - XXI

IMMUNOHAEMATOLOGY & BLOOD TRANSFUSION

DEPARTMENT OF TRANSFUSION MEDICINE

THE TAMILNADU Dr.M.G.R. MEDICAL UNIVERSITY

CHENNAI, INDIA

MAY 2020

ABSTRACT

Background and Aim: Transfusion-transmissible infections such as HIV, HCV and HBV are among the greatest threats to blood safety and potential serious chronic sequelae associated with readily transmitted agents. The implementation of these new highly sensitive 4th generation ELISA tests has significantly enhances early detection of HIV,HCV and HBV infection compare with 3rd generation ELISA.Hence, this study is aimed to compare the 4th generation ELISA with third generation ELISA for early detection of HIV, HCV and HBV infection among voluntary blood donors.

Materials and Methods: This Cross-sectional study was conducted over one year period from August 2018- July 2019 in the Department of Transfusion Medicine, The Tamil Nadu Dr.MGR Medical University, Guindy, Chennai. All blood units were tested for HIV I&II, HCV antibodies and HBsAg using

3rd generation ELISA (Merilisa hiv 1 & 2 gen 3, Merilisa HCV and Merlisa HBsAg kits manufactured by Meril diagnostics). In addition donor units were screening with 4th generation HIV Ag-Ab, HCV Ag-Ab and HBsAg ELISA (Genscreen ULTRA HIV Ag-Ab kits, Monolisa HCV Ag-Ab ULTRA V2 kit and Monolisa HBsAg ULTRA ELISA kits manufactured by Bio Rad).

Results: Among total 373 voluntary blood donor samples, 3 were found to be seroreactive for HBsAg by 3rd and 4th generation ELISA. Only one sample was found to be seroreactive for HCV by 4th generation ELISA, this was found to nonreactive by 3rd generation ELISA. None of the samples were reactive for HIV by both 3rd and 4th generation ELISA.

Conclusion: In the present study, there is no difference in detection of HBsAg and HIV between 3rd and 4th generation ELISA. In detection of HCV by 4th generation ELISA is found to be more sensitive than 3rd generation ELISA.

TABLE OF CONTENTS

SL.NO TITLE PAGE NO 1 INTRODUCTION 1 2 AIM AND OBJECTIVE 4 3 REVIEW OF LITERATURE 5 4 MATERIALS AND METHODS 52 5 RESULTS 72 6 DISCUSSION 94 7 SUMMARY 99 8 CONCLUSION 100 9 BIBILIOGRAPHY i-xvii 10 ANNEXURES Ethical Committee Clearance Documents Plagiarism Clearance Document Donor Information Sheet Donor Consent Form Study Questionnaire Master Chart

LIST OF ABBREVIATION

AABB American Association of Blood Banks CDC Centers for Disease Control and Prevention CD Cluster of Differentiation CCR5 C-Chemokine Receptor 5 DGHS Directorate General of Health Services DNA Deoxyribonucleic Acid ELISA Enzyme Linked Immunosorbent Assay FDA Food and Drug Administration gp Glycoprotein HIV Human Immunodeficiency Virus HBV Hepatitis B Virus HCV Hepatitis C Virus HBsAg Hepatitis B Surface Antigen IgM Immunoglobulin M IgG Immunoglobulin G NAT Nucleic acid Amplification Test OD Optical Density ORF Open Reading Frame RNA Ribonucleic acid RT Reverse Transcriptase VDRL Venereal Disease Research Laboratory Test TMB Tetra Methyl Benzidine TTI Transfusion Transmitted Infections WHO World Health Organization

INTRODUCTION

1. INTRODUCTION

According to World Health Organisation (WHO), blood donation should be absolutely voluntary, non-remuneration and the motive should be purely altruistic to help the unknown recipient.1

Blood Safety, thus, remains an issue of major concern in transfusion medicine in developing countries.

WHO supports countries in developing national blood systems to ensure timely access to safe and sufficient supplies of blood and blood products by following good transfusion practices to meet the patient’s needs.8

The programme encourages to work towards self-sufficiency in safe blood and blood products based on voluntary unpaid blood donation. It provides policy guidance and technical assistance to ensure universal access to safe blood and blood products, thereby achieve universal health coverage.

WHO, in addition to promoting unpaid voluntary blood donation, recommends all donations should be mandatorily screened for highly prevalent infections which are likely to be transmitted by blood transfusion.

In India, it is mandatory to test every unit of blood collected for Hepatitis

B, Hepatitis C, HIV, Syphilis and Malaria. If a donor is found to be positive to any of these five infections, their blood is considered infectious and discarded.10

The scrupulous screening for these infections is all the more important because the blood units donated by apparently healthy and asymptomatic blood donors carry infectious agents with them.2

1 In order to improve the standards of Blood and its components, the

Central Govt. through Drugs Controller General of India, has formulated a comprehensive legislation to ensure better quality control system on collection, storage, testing and distribution ofblood and its components. Central Govt. amended from time to time the existing requirements of Blood Banks in the Drugs &

Cosmetics Act, 1940 and Rules there under 1945 to meet the latest standards.44

Transfusion-transmissible infections such as HIV, HCV and HBV are among the greatest threats to blood safety and potential serious chronic sequelae associated with readily transmitted agents.20

Screening of blood donors first started in 1947.21Government of India has made mandatory to screen donated blood for HBV (since 1971), HIV (since

1989) and HCV (since 2001).3,4,5

The risks of giving blood during infectious window period were estimated as follows: for HIV, 1 in 4,93,000; for HCV, 1 in 1,03,000; and for HBV, 1 in 63,000.7In NACO guidelines83 2016 seroprevalence among blood donors in India for HIV is 0.26%, HCV 0.4 to 2 % and HBV 2 to 3%.

Nucleic acid testing (NAT) is a molecular technique for screening blood donations to reduce the risk of Transfusion Transmitted Infections (TTIs) in the recipients, thus providing an additional layer of blood safety. It was introduced in the developed countries in the late 1990s and early 2000s and presently around 33 countries in the world have implemented NAT for human immunodeficiency virus

(HIV) and around 27 countries for hepatitis B virus (HBV).15

In India, mandatory blood screening for HBV, HIV and HCV is done by serological tests for HBsAg and antibodies to HIV 1&2 and HCV. The screened

2 seronegative donations are still at risk for TTIs and thus, need for a sensitive screening test arises to decrease this residual risk which has been reduced significantly over the last two to three decades in western countries where NAT has been implemented. NAT testing has been started in few centres in India, but it is not a mandatory screening test for TTIs as per Drug and Cosmetics Act, 1940 and the rules therein.16

Major barriers in implementing routine NAT testing in India are its high cost and lack of technical expertise in most of the blood centers.14

In India, currently using 3rd generation ELISA KITS which will detect either antigen or antibody. The 4th generation ELISA assays simultaneously detect antibodies against HIV-1 and 2 and the presence of p24 antigen and thus shorten the window period to about 14 days, as compared to about 22 days with 3rdgeneration,6 for HCV simultaneous detection of Capsid Antigen and antibodies against NS1 to

NS5 shortens the window period to about 10 to 20 days,22,134as compared to 59 days with 3rd generation ELISA, for HBV ultrasensitive kit detection of various subtypes of HBsAg and the most part of variants HBV strains thus detect in the window period to about 24 days, as compared 37 days with 3rd generation ELISA.22 The implementation of these new highly sensitive 4th generation ELISA tests has significantly enhances early detection of HIV,HCV and HBV infection.6

Hence, this study is aimed to compare the 4th generation ELISA with third generation ELISA for early detection of HIV, HCV and HBV infection among voluntary blood donors.

AIM AND OBJECTIVE

2. AIM ANDOBJECTIVES

x To compare the sensitivity of third and fourth generation ELISA for

HIV, HBV and HCV screening of blood units collected from

voluntary blood donors. x To find out the specificity of the seroreactive samples will be

confirmed by RT-PCR.

REVIEW OF

LITERATURE

3. REVIEW OF LITERATURE

Blood Transfusion Safety

In 1975, 28th World Health Assembly in Geneva passed a resolution to address the issue of blood safety. The resolution WHA 28.72 urges member states for the development of national blood services on voluntary non remunerated donation of blood.47 The aim of Blood Transfusion safety is to ensure that everyone has access to blood and blood products which are as safe as possible, adequate to meet the need of patients, available at reasonable cost, transfused only when necessary and provides us a sustainable blood program within in the health care system.46

Introduction to the global framework85

This global framework for action to achieve 100% voluntary blood donation has been developed jointly by the World Health Organization and the

International Federation of Red Cross and Red Crescent Societies. It is designed to provide guidance and support to countries seeking to establish effective voluntary blood donor programmes, phase out family/replacement blood donation and eliminate.

Voluntary blood donation (VBD)

VBD are the cornerstone of a safe and adequate supply of blood and blood products. The safest blood donors are voluntary, non-remunerated blood donors from low-risk populations. Despite this notion, family/replacement donors still provide more than 45% of the blood collected in India. Such donors are supposed to be associated with a significantly higher prevalence of transfusion-

5 transmissible infections (TTIs) including Human immune deficiency virus (HIV), hepatitis C virus(HCV) hepatitis B virus (HBV), syphilis and malaria.17

It had been known for some time that blood transfusion transmits syphilis, malaria and smallpox. In 194245, VDRL test was the first screening test to be made mandatory in U.S, since there was a strong suspicion of transmission of syphilis by blood transfusion.

It was during 1953, Paul Beeson, described transfusion transmitted hepatitis. Subsequently they found Hepatitis A, B, and C to be transmitted through blood transfusion. However, it was the outbreak of AIDS that galvanized the public attention to blood transfusion.

In the year 1985, HIV screening was made mandatory in U.S blood banks. Improved donor screening and increased testing have greatly decreased the risk of disease transmission and rendered the blood supply safer.

The disease transmission depends on the type of donor, their behavior and risk status.

The following are the various types of donors:

1. VOLUNTARY DONORS

A person who gives blood, plasma or other blood components of his /her own free will and receives no payment for it, either in the form of cash or in kind which could be considered a substitute for money, this includes time off work other than reasonably needed for the donation and travel. Small tokens, refreshments and reimbursement of the direct travel costs are compatible with voluntary non-

6 remunerated blood donation. He is an altruistic donor, who gives blood fully andwillingly.48

CATEGORIES OF VOLUNTARY BLOOD DONOR: a) New voluntarydonor:

A Voluntary non-remunerated blood donor who has never donated blood

before b) Lapsed voluntary donor: first write regular donor and thenlapsed

A Voluntary non- remunerated blood donor who has given blood in the

past but does not fulfill the criteria for a regular donor c) Regular voluntary donor :

A Voluntary non-remunerated blood donor who has donated at least three

times, the last donation being within the previous year and continuous to

donate regularly at least once per year.

2. OTHER CATEGORIES OF BLOODDONORS

1) FAMILY / REPLACEMENT BLOODDONOR:

a) A donor who gives blood when it is required by a member of the

patients’ family or community. This may involve a hidden paid

donation system in which the donor is paid by the patient’s family.

b) A family / replacement donor is one who gives blood when it is

required by a member of his/her family or community. This often

7 involves concern and or payment which compromise the safety of

theblood.

c) A member of the family or a friend of the patient who donates blood

in replacement of blood needed for the particular patient without

involvement of any monetary or other benefits from anysource.

2) PAID / PROFESSIONALDONOR:

A donor who donates blood in exchange of money or other forms of payment is called a professional donor. It is banned in India from 1st Jan 1998.48

Many reports confirm that paid donors and replacement donors have a higher prevalence of TTI. In a study conducted by Gupta et al (New Delhi) in 2006 among armed forces, seropositivity of HIV, anti-HCV, HBsAg show an increase in reactivity rate in replacement donor compared to voluntary donors.49

Every year blood transfusion rescues millions of lives worldwide, recipients of transfusions risk becoming infected with blood-borne pathogens. TTIs agents like HIV, HCV and HBV infections are one of the major medical concerns in the contemporary world.9

WHO data states the current prevalence of TTIs in blood donors for lower-middle income countries like India to be 1.60%, 0.20% and 0.40% for HBV,

HIV and HCV respectively.8 The prevailing screening tests available for TTI testing are Enzyme Linked Immuno-Sorbent Assay (ELISA), chemiluminescence test, nucleic acid amplification test etc. Inspite of the above stated screening tests, there are residual risks that may cause transmission of infections to the recipients.9

8 HIV

HIV-1 or HIV-2, which are retroviruses in the Retroviridae family, Lentivirus genus. HIV is different in structure from other retroviruses. It is roughly spherical11 with a diameter of about 120 nm, around 60 times smaller than a red blood cell.12 It is composed of two copies of positive-sense single- strandedRNA that codes for the virus's nine genes enclosed by a conical capsid composed of 2,000 copies of the viral protein p24.13 The single- stranded RNA is tightly bound to nucleocapsid proteins, p7, and enzymes needed for the development of the virion such as reverse transcriptase, proteases, ribonuclease and integrase. A matrix composed of the viral protein p17 surrounds the capsid ensuring the integrity of the virion particle.13

HIV STRUCTURE

Figure: 1 Structure of HIV

9 Comparison of HIV species

Species Virulence Infectivity Prevalence Inferred origin

HIV-1 High High Global Common chimpanzee

HIV-2 Lower Low West Africa Sooty mangabey

Table: 1 Comparison of HIV species

The RNA genome consists of at least seven structural landmarks, nine genes and encoding 19 proteins.

HIV proteins and function24

Gene Size* Protein Function gag Pr55Gag precursor of the inner structural proteins p24 capsid protein (CA) formation of conical capsid myristilated protein, forming the inner p17 matrix protein (MA) membrane layer formation of the nucleoprotein/RNA p7 nucleoprotein (NC) complex p6 involved in virus particle release pol Pr160GagPol precursor of the viral enzymes proteolytic cleavage of Gag (Pr55) and Gag- p10 protease (PR) Pol (Pr160GagPol) precursor protein; release of structural proteins and viral enzymes reverse transcriptase p51 transcription of HIV RNA in proviral DNA (RT) degradation of viral RNA in the viral p15 RNase H RNA/DNA replication complex integration of proviral DNA into the host p32 integrase (IN) genome

10 Gene Size* Protein Function precursor of the envelope proteins SU and PrGp160 TM, cleavage by cellular protease attachment of virus to the target cell env surface glycoprotein gp120 anchorage of gp120, fusion of viral and cell (SU) transmembrane gp41 membrane protein (TM) tat p14 transactivator protein activator of transcription of viral genes regulates the export of non-spliced and rev p19 RNA splicing regulator partially spliced viral mRNA myristilated protein, influence on HIV negative regulating replication, enhancement of infectivity of nef p27 factor viral particles, downregulation of CD4 on target cells and HLA cells on target critical for infectious virus production in vif p23 viral infectivity protein vivo component of virus particles, interaction vpr p15 virus protein r with p6, facilitates virus infectivity, effect on the cell cycle efficient virus particle release, control of vpu p16 virus protein unique CD4 degradation, modulates intracellular trafficking interaction with p6 in virus particles, vpx p15 virus protein x involved in early steps of virus replication of HIV-2, component of virus particles Tat-Env-Rev fusion protein, regulates the tev p26 tat/rev protein activity of Tat and Rev in nucleus

Table: 2 HIV proteins and function

Figure: 2 Genome layouts of HIV 1 & 2

Tropism

The term viral tropism refers to the cell types a virus infects. HIV can infect a variety of immune cells such as CD4+ T cells, macrophages, and microglial cells. HIV-1 entry to macrophages and CD4+ T cells is mediated through interaction of the virion envelope glycoproteins (gp120) with the CD4 molecule on the target cells' membrane and also with chemokine co-receptors.18,19

Macrophage-tropic (M-tropic) strains of HIV-1, or non-syncytia- inducing strains (NSI; now called R5 viruses85 use the ȕ-chemokine receptor, CCR5, for entry and are thus able to replicate in both macrophages and CD4+ T cells.84

Macrophages and microglial cells are the cells infected by HIV in the central nervous system. In the tonsils and adenoids of HIV-infected patients, macrophages fuse into multinucleated giant cells that produce huge amounts of virus.

12 T-tropic strains of HIV-1, or syncytia-inducing strains (SI; now called

X4 viruses)85 replicate in primary CD4+ T cells as well as in macrophages and use the Į-chemokine receptor, CXCR4, for entry. 23,84,91

Dual-tropic HIV-1 strains are thought to be transitional strains of HIV-1 and thus are able to use both CCR5 and CXCR4 as co-receptors for viral entry.

HIV-2 is much less pathogenic than HIV-1 and is restricted in its worldwide distribution to West Africa. The adoption of "accessory genes" by HIV-2 and its more promiscuous pattern of co-receptor usage (including CD4- independence) may assist the virus in its adaptation to avoid innate restriction factors present in host cells. A survival strategy for any infectious agent is not to kill its host, but ultimately become a commensal organism. Having achieved a low pathogenicity, over time, variants that are more successful at transmission will be selected.31

Mechanism of viral entry:25

1. Initial interaction between gp120 and CD4.

2. Conformational change in gp120 allows for secondary interaction with CCR5.

3. The distal tips of gp41 are inserted into the cellular membrane.

4. gp41 undergoes significant conformational change; folding in half and forming coiled-coils.

Replication and transcription

Shortly after the viral capsid enters the cell, an enzyme called reverse transcriptaseliberates the positive-sense single-stranded RNA genome from the

13 attached viral proteins and copies it into a complementary DNA (cDNA) molecule.37. The cDNA and its complement form a double-stranded viral DNA that is then transported into the cell nucleus. The integration of the viral DNA into the host cell's genome is carried out by another viral enzyme called integrase.37

During viral replication, the integrated DNA provirus is transcribed into

RNA, some of which then undergo RNA splicing to produce mature messenger

RNAs (mRNAs). These mRNAs are exported from the nucleus into the cytoplasm, where they are translated into the regulatory proteins Tat (which encourages new virus production) and Rev. As the newly produced Rev protein is produced it moves to the nucleus, where it binds to full-length, unspliced copies of virus RNAs and allows them to leave the nucleus.40

Some of these full-length RNAs function as new copies of the virus genome, while others function as mRNAs that are translated to produce the structural proteins Gag and Env. Gag proteins bind to copies of the virus RNA genome to package them into new virus particles.41

HIV-1 and HIV-2 appear to package their RNA differently.42 HIV-1 will bind to any appropriate RNA. HIV-2 will preferentially bind to the mRNA that was used to create the Gag protein itself.43

Figure:3 HIV RNAReplication and transcription

Infection:

HIV infection can be transmitted through sexual contact, in utero or childbirth, breastfeeding, and parenteral exposure to blood. The CDC reported that in the United States in 2015, the highest incidence of new diagnosis was in African-

Americans, males, age 20–29 years old and the most common risk identified was male-to-male sex. Rate per 100,000 of new infections among women is dropped from 7.3% in 2010 to 5.4% in 2015. In comparison, among men, rate dropped from

27.3% in 2010 to 24.4% in 2015.27

60% of acute HIV infections result in nonspecific flulike illness with incubation period of 2–4 weeks. Acute infection resolves in weeks to few months resulting in asymptomatic period that may last years. Eventually, the virus can no

15 longer be controlled, as helper CD4+ (T4) lymphocytes are destroyed. This loss of

CD4+ lymphocytes results in development of opportunistic infections and direct viral effects on multiple organs; together resulting in death on average after 3 years once AIDS is diagnosed, if not treated. Post infection survival has improved with the advent of potent antiretroviral therapies. However, these medications do not eradicate HIV and have multiple side effects. Moreover, resistant viral strains have been known to develop, which adds to the difficulty of treatment.

Figure:4CD34+ COUNT VS HIV RNA COPIES

HBV

Transmission of hepatitis through blood transfusion was first reported in

1943 by Beeson.52 Hepatitis transmitted through faecal-oral mechanisms was termed hepatitis A (‘epidemic hepatitis’), while hepatitis transmitted through blood and its derivatives (‘serum hepatitis’) was termed hepatitis B. These terms were formally adopted by the World Health Organization in the 1970s.53

16 Prevention of the transmission of hepatitis by blood transfusions began with donor screening through the use of a donor questionnaire, primarily for a history of hepatitis, commonly referred to as ‘yellow jaundice’. Laboratory screening for hepatitis B virus began in 1969, with the testing of blood donors for what eventually became known as the hepatitis B surface antigen (HBsAg).

HBV was the first human hepatitis virus identified and discovered in

1963 by Blumberg,50Initially termed the Australia antigen, due to its original description in an Australian aborigine, and in 1965 linked to leukaemia, HBV was eventually noted to have a high prevalence in multiply transfused patients.50,51

HBV GENOME

Figure: 5 HBV GENOME

17 HBV infection pathomechanism

Molecular Virology Hepatitis B virus is a 42-nm DNA virus composed of an outer shell (7 nm in width) and an inner core (27 nm in diameter).54-56

It belongs to a novel genus, Hepadnavirus, within the Hepadnaviridae family; HBV is designated hepadnavirus type 1.

The outer coat material (or envelope), is composed of three virus- encoded polypeptides, collectively designated the hepatitis B surface antigen, and host derived lipid components. The HBV envelope proteins can be detected in serum, not only as a part of the intact virus, but also as separate 22-nm spherical or tubular sub virus particles,56 that circulate in amounts that outnumber complete HBV particles by a factor of 104 , that do not contain DNA, and that are highly immunogenic, but non-infectious.

They, in fact, constituted the source for the first generation of plasma- derived HBV vaccines. Clinically, HBsAg is detected in serum by serologic assays, visualized in the cytoplasm of hepatocytes by electron microscopy, and identified in liver tissue of infected persons by its ground glass appearance and by antigen- specific immunohistochemical staining. The inner icosahedral nucleocapsid core of

HBV is composed of the nucleocapsid protein known as the hepatitis B core antigen

(HBcAg), the viral genome, and the viral DNA polymerase. The genome of HBV is a relaxed circular, partially double-stranded DNA, with a complete negative DNA strand (3200 nucleotides) and an incomplete positive DNA strand.

The genomic information of the virus, present on the long strand of the

DNA, comprises four highly overlapping (67%) open reading frames (referred to as

18 S, C, P, and X genes), encoding the envelope (pre-S/S), core (precore/core), polymerase, and X proteins, respectively (Fig: 6).55,56,58

The S gene has three in-frame start codons and a common stop codon, dividing the gene into the preS1, preS2, and S regions encoding the large (L), middle (M), and small (S) envelope proteins, respectively, depending on where translation is initiated. The product of the S region is the HBsAg (major protein).57

The M and S envelope proteins are found in both virions and subvirus particles, whereas the L proteins are found mostly in complete virions. The C gene has two in-frame start codons. The core protein (HBcAg) is translated from the second start codon and expressed on the surface of the nucleocapsid.

Figure: 6 HBV genomic information

19 Translation from the precore start codon produces a precore polypeptide that is posttranslationally cleaved into a soluble nonparticulate protein known as the hepatitis B e antigen (HBeAg).

The P gene, which encompasses 80% of the viral genome, encodes a single multifunctional protein with four distinct domains: a protein primer, a spacer, a reverse transcriptase (RT)/ DNA polymerase, and an RNAse H domain. The X gene encodes a multifunctional transactivator of transcription (HBxAg), of both viral and cellular genes.59,60

It has pleiotropic effects on the transcriptome of the infected cell, including signalling pathways, protein degradation, cell cycle checkpoints, cell death, and carcinogenesis. The HBV genome contains four promoters (preS1, preS2/S, C and X) that control transcription by the cell RNA polymerase II of six functionally co-terminal viral mRNAs.61

HBV REPLICATION

Infects liver cells by the bloodstream, begins with attachment of the virion to the hepatocyte membrane. Sodium taurocholate co-transporting polypeptide (NTCP) receptors, which binds to the pre-S1 region of the HBV envelope.62

The virion is uncoated in the hepatocyte cytoplasm and the viral genome enters the hepatocyte nucleus.Formation of cccDNA by Cellular repair enzymes,cccDNA activates the cellular gene expression producing four mRNA.63

The mRNA then moves to the cytoplasm and is translated into protein.

This mRNA work as matrix for core protein and DNA polymerase. Self-assembly of

20 core protein, DNA polymerase together and embed the mRNA.DNA polymerase which transcribes the core particles of RNA into DNA, after producing DNA plus strand the RNA is cleaved.

The residual 185´ terminal bases of the RNA pre-genome represent the primer for the plus strand.In parallel to the genome maturation, sub genomic mRNA molecules [3-DO-carboxy-terminal HBs proteins, S(small), M(middle), and

L(large), with preS1, preS2, and S protein structures] are expressed.

The HBs proteins are released into the endoplasmic reticulum within the endoplasmic reticulum; the HBs particle embeds the circlet core particle and builds the final HB-virus.The HBsAg as well as the HB virus are released via Golgi apparatus into the bloodstream.64

HBV REPLICATION

Figure: 7 HBV replication

21 HBV Transmissions65

x Infected patients contain HBV virus in blood, saliva, semen, breast milk, vaginal, and sore secretions.

x Vertical infection from mother to child.

x Heterosexual or homosexual contacts are reported.

x Contaminated, inefficiently sterilized medical devices.

x Personal devises such as tooth brushes, razors.

x Thorns and branches accidentally contaminated with blood have transmitted HBV.

x Fomites such as finger-stick devices used to obtain blood for glucose measurements, multi-dose medication vials, jet gun injectors, and endoscopes.

x HBV can survive on counter tops for seven days and remains capable of causing infection during that time period.

x Contaminated file cards among laboratory technicians

x In dialysis units,- common-dose medications and shared equipment

Genetic Variation of HBV

HBV can be classified into eight genotypes (A to H), The prevalence of specific genotypes varies geographically, with genotype A found mainly in Northern Europe, North America, India, and Africa; genotypes B and C in Asia; genotype D in Southern Europe, the Middle East, and India. 67

All known genotypes have been found in the United States, with prevalence of genotypes A, B, C, and D of 35%, 22%, 31%, and 10%, respectively,

22 while genotypes E to H represent 2%.66 Most common and clinically relevant HBV mutants have been well characterized and affect the precore/core promoter, preS/S, and polymerase genes.67

Precore and Core Promoter Variants

In the United States, precore variants have been found in 27% of patients with chronic HBV infection.66 The most common core promoter variant involves a dual mutation A1762T, G1764A (TA change).

In the United States, these variants have been found in 44% of patients with chronic HBV infection, and may also be associated with HBeAg-negative CHB.39HBeAg-negative CHB has been estimated to account for 7% to 30% of patients with CHB worldwide, being predominant in Mediterranean countries and Asia.68,69

The most common HBV variant associated with HBeAg-negative CHB, G1896A, usually occurs in HBV genotype D infection (more prevalent in the Mediterranean basin)

Pre-s/s Variants

HBV S gene mutants have been described in infants infected with HBV despite an adequate anti-HBs response to HBV vaccination (vaccine escape mutants).

The most common mutation involves an amino acid substitution at codon 145 (G145R) in the “a” determinant of HBsAg. Recurrent hepatitis B after liver transplantation despite HBIG prophylaxis has also been associated with this mutation.70

23 Marker and Characteristics of HBV

Marker Characteristics HBsAg71 • First serological marker appear in HBV infection • Window period between HBV infection and detection of HBsAg estimated to be around 38 days, but depends on analytical sensitivity of assay used. • Quantification of HBsAg is a potential alternative marker of viraemia and to monitor response to antiviral treatment Anti-HBc • High levels present during acute infection but may remain IgM72 detectable for up to 6 months. • Used to differentiate between acute and chronic HBV infection, but its reappearance during “flares” in chronic HBV infection make it an unreliable indicator of recent primary HBV infection. Anti-HBc • Develops around 3 months after infection and most constant (total)72 marker of infection • Together with anti-HBs, indicates resolved infection • Anti-HBc, with or without anti-HBs, also indicates individuals who may reactivate in the context of immunosuppression HBeAg71 • Present when the virus is actively replicating in the liver. • Associated with high levels of HBV viraemia and is therefore a marker of “high infectivity” and association with progressive liver disease. Anti-HBe73 • Represents host response and indicates decreasing HBV DNA and therefore infectivity • May coexist with HBeAg during the period of seroconversion at the end of immune-tolerance phase Anti-HBs73 • Neutralizing antibody that confers protection from infection. • Generated by immunization and used to monitor post- immunization responses (anti-HBc absent). HBV DNA73 • Used as a more direct and accurate measure of active HBV viral replication, which correlates with disease progression • Serum HBV DNA is measured in international units (IU)/mLd as the recognized international standard or copies/ml by nucleic acid testing (NAT) technologies. Table:3 Marker and Characteristics of HBV

Figure: 8 HBV Markers

Figure: 9 HBV serological test results and interpretation

HCV

The hepatitis C virus (HCV) is a small, enveloped, single-stranded, positive-sense RNA virus.75

25 It is a member of the genus Hepacivirus in the family

Flaviviridae.81There are seven major genotypes of HCV, which are known as genotypes one to seven.82 The genotypes are divided into several subtypes with the number of subtypes depending on the genotype.

In the United States, about 70% of cases are caused by genotype 1, 20% by genotype 2 and about 1% by each of the other genotypes.80 Genotype 1 is also the most common in South America and Europe.75

It was known that NANB hepatitis was associated with intravenous drug use, treatment with pooled plasma derivatives before viral inactivation processes were implemented and transfusions prior to 1990.

After the cloning of its genome in 1989, 86 NANB hepatitis, now termed hepatitis C virus (HCV), was determined to be a major aetiological agent of chronic viral hepatitis worldwide. A recent systematic review estimated that 110 million persons have a history of HCV infection (i.e. are HCV-antibody positive) and 80 million have chronic viraemic infection.88

Regions estimated to have a high prevalence in the general population

(>3.5%)are Central and east Asia, and North Africa/Middle East; those with a moderate prevalence (1.5–3.5%).88 Updated estimates in Africa show a HCV prevalence of 2.98%, with a higher prevalence observed in west Africa and lower in south-east Africa.89

An estimated 143 million people (2%) worldwide are infected with hepatitis C as of 2015.76 In 2013 about 11 million new cases occurred.78 It occurs most commonly in Africa and Central and East Asia.74 About 167,000 deaths due to

26 liver cancer and 326,000 deaths due to cirrhosis occurred in 2015 due to hepatitis

C.77

The existence of hepatitis C is originally identifiable only as a type of non-A non-B hepatitis –was suggested in the 1970s and proven in 1989.79

Hepatitis C virus is a single-stranded, RNA, enveloped (lipid coated) virus in the viral family Flaviviridae. This lipid coat can be destroyed by solvent/detergent, leading to HCV inactivation.

Hepatitis C virus is transmitted primarily through contact with blood.

Intravenous drug use is the major risk behaviour. Before anti-HCV testing was available, blood transfusion recipients were at high risk for HCV infection.

Historically, testing for elevated transaminases during blood donation was a surrogate test for NANB hepatitis in donor blood, but as shown retrospectively, along with anti-HBc testing in theUSA, surrogate testing had little impact on HCV transmission.87

HCV Replication

In HCV infection, the virus travels via the blood to the primary target cell, the hepatocyte. Once in the liver, the virus attaches to the receptor (CD81)and enters the hepatocyte cytoplasm.92

In the cytoplasm, the viral RNA is translated into proteins using host rough endoplasmic reticulum. Nascent RNA is then made from negative strand viral

RNA (unlike HBV, which uses DNA and a reverse transcriptase).92

27 HCV genome

Characterization of HCV shows that the viral genome consists of single- stranded, linear RNA of 9.6 kb that is positively stranded and hence can serve directly as message for the synthesis of viral proteins.93, 94

The viral proteins are encoded from a single, large open reading frame that produces a polyprotein that is posttranslationally cleaved.94

The HCV genome is characterized by a highly conserved, untranslated region at the 5 terminus (5UTR) that is now used as the primary target for molecular amplification assays to detect HCV RNA and for viral genotyping.

The 5UTR contains an internal ribosomal entry site that is essential for viral protein synthesis and that initiates translation at the start codon of the large open reading frame.94 Downstream of the 5UTR are the coding regions for envelope proteins designated E1 and E2, followed by the coding region for the viral core (nucleocapsid).

In the terminal portion of the E1 region is a hyper variable segment (HVR1), which is the most highly mutable region of the genome, presumably because its position in the viral envelope places it under a high degree of immune pressure.

It is presumed that this region is the target for neutralizing antibody responses. Downstream from the structural genomic regions are a series of non- structural coding regions designated NS2, NS3, NS4, and NS5.92

The full function of these regions is not known, but they have been shown to code for critical enzymes, including an NS3 protease, an NS3 helicase, and an NS5 polymerase.

28 HCV GENOME

Figure: 10 HCV Genome

HCV REPLICATION

In HCV infection, the virus travels via the blood to the primary target cell, the hepatocyte. Once in the liver, the virus attaches to the receptor (CD81)and enters the hepatocyte cytoplasm.92

In the cytoplasm, the viral RNA is translated into proteins using host rough endoplasmic reticulum. Nascent RNA is then made from negative strand viral

RNA (unlike HBV, which uses DNA and a reverse transcriptase).92

There are six major genotypes of HCV that are designated in Arabic numerals 1 through 6 and each has been subtyped as designated by lowercase lettering.

The genotypes differ from each other in nucleotide sequence by at least

15%. Genotypes 1a and 1b are the most prevalent forms in the United States,

Europe, and Japan. In the United States, genotypes 1a and 1b are found in almost equal frequency, whereas in Japan genotype 1b predominates.95, 96

29 HCV Replication

Figure:11 HCV Replication

Genotypes 2 and 3 are found as minor populations in the United States

(10% genotype 2 and 6% genotype 3) and Europe.96, 97 Genotype 3 is the predominant genotype in Southeast Asia. Genotype 4 appears isolated to Northern

Africa (especially Egypt), and the Middle East, whereas genotype 5 has been localized to South Africa. Genotype 6 is rare and found predominantly in Japan.96, 98

Biochemical, serologic, and molecular biologic profile of acute and chronic transfusion-associated HCV infection.

The magnitude of HCV RNA elevation as measured by means of branched DNA assay, the interval to the first elevation in the level of alanine aminotransferase (ALT), the magnitude of ALT elevation in the acute phase, or the interval between exposure and the first appearance of antibody.99

30 Progression to chronic disease cannot be predicted in the acute phase, and the only distinguishing features in these patterns are the persistence of ALT elevation and the persistence of HCV RNA in persons with chronic hepatitis C.100

The major peak of viral replication (assessed with HCV RNA level) occurs before the first increase in ALT level and before any clinical or biochemical evidence of hepatitis; it is presumed that persons might be most infectious in this interval before the acute phase.100, 101

In acute resolving infection, HCV RNA levels increase rapidly before the decline in serum ALT level.

Screening assays

TYPES OF ASSAY

Various types of assay have been developed for use in blood screening over the past three decades. However, not all assays are suitable in all situations and each assay has its limitations which need to be understood and taken into consideration when selecting assays.102

The main types of assay used for blood screening are:

Immunoassays (IAs)

- Enzyme immunoassays (EIAs)

- Chemiluminescent immunoassays (CLIAs)

- Haemagglutination (HA)/particle agglutination (PA) assays

- Rapid/simple single-use assays (rapid tests)

Nucleic acid amplification technology (NAT) assays

31 Immunoassays102

Immunoassays are assay systems available in several formats that may be used to detect antibody, antigen or a combination of the two. Commonly used antigen detection assays are based on the use of immobilized antibody to capture pathogen-specific antigens present in the sample.

Immunoassays can be used in different situations from high through-put laboratories with full automation to medium-sized laboratories with semi- automation to small laboratories, such as those in remote areas, which conduct a small number of tests manually.

Enzyme immunoassays (EIAs) and chemiluminescent immunoassays (CLIAs) 102

Enzyme and chemiluminescent immunoassays are currently the most commonly used assays for screening donated blood for TTIs. The design of EIAs and CLIAs is similar and they differ only in the mode of detection of immune complexes formed – colour generation in EIAs and measuring light produced by a chemical reaction in CLIAs.

Any of these types of IA with high sensitivity will generally detect the target markers of infection required if they have been properly evaluated for blood screening and are then used within a quality environment.

EIAs and CLIAs are suitable for the screening of large numbers of samples and require a range of specific equipment. These assays may be performed either manually or on non-dedicated automated assay processing systems (open system). They may also be manufactured specifically to operate on specific dedicated automated systems (closed system).

Particle agglutination assays102

32 Particle agglutination assays detect the presence of specific antibody or antigen in a test sample through the agglutination of particles coated with the complementary specific antigen or antibody respectively.

Agglutination assays, mainly antibody assays, use a range of particles including red cells (haemagglutination) and inert particles such as gelatin and latex.

This use of inert particles has the advantage of reducing non-specific reactivity against cross-reacting red cell antigens. The basic principles of haemagglutination and particle agglutination assays are the same, irrespective of the type of particles used. PA assays are still used extensively for the detection of syphilis antibodies.

Rapid/simple single-use assays (rapid tests) 102

Rapid/simple single-use assays are discrete, individual, disposable assays: i.e. they are used once and discarded. These assays exist in a number of different presentations. Many rapid tests are based on a form of immunochromatography in which the added sample flows down an inert strip and reacts with previously immobilized reagents. The sample can be serum, plasma or even whole blood in some cases.

Nucleic acid amplification technology assays

Nucleic acid amplification technology (NAT), as applied to blood screening, detects the presence of viral nucleic acid, DNA or RNA, in donation samples. In this technology, a specific RNA/DNA segment of the virus is targeted and amplified in-vitro. The amplification step enables the detection of low levels of virus in the original sample by increasing the amount of specific target present to a level that is easily detectable. The presence of specific nucleic acid indicates the presence of the virus itself and that the donation is likely to be infectious.

33 NAT assays can either be performed on individual donations (ID) or on mini-pools (MP) to detect the nucleic acid of the infectious agent. In addition to

NAT assays which target individual viral nucleic acids, multiplex NAT screening assays have been developed which can detect DNA or RNA from multiple viruses simultaneously.

SELECTION OF ASSAYS

The selection of appropriate assays is a critical part of the screening programme. Reliable results depend on the consistent use of well-validated and effective assays. A number of factors need to be considered in selecting the most appropriate assays. In general, a balance has to be found between screening needs and the resources available, including finances, staff and their expertise, equipment, consumables and disposables.

Each screening system has its advantages and limitations that should be taken into consideration when selecting assays. Some limitations include:

The length of time following infection before the screening test becomes reactive (window period)

Rates of biological false positives which may result in the wastage of donations and unnecessary deferral of donors

The complexity of some systems that require automation.

CRITICAL ASSAY CHARACTERISTICS

Sensitivity and specificity are the key factors to be considered in selecting a specific assay. For the screening of blood donations, both sensitivity and specificity should be the highest possible or available. Each assay should be

34 evaluated within the country or region to confirm the technical data provided with regard to assay performance and where possible, data from other studies should be analysed.

ELISA

• EL Enzyme-linked

• IS Immunosorbent

• A Assay

Before the development of the ELISA, the only option for conducting an immunoassay was radioimmunoassay, a technique using radioactively labelled antigens or antibodies. Radioimmunoassay was first described in a scientific paper by Rosalyn Sussman Yalow and Solomon Berson published in 1960.103

A suitable alternative to radioimmunoassay would substitute a nonradioactive signal in place of the radioactive signal because radioactivity poses a potential health threats.

Principle

ELISA is an analytical biochemistry assay and a "wet lab" technique, it involves detection of an analyte (i.e., the specific substance whose presence is being quantitatively or qualitatively analyzed) in a liquid sample by a method that continues to use liquid reagents during the analysis.104,105

Controlled sequence of biochemical reactions that will generate a signal which can be easily quantified and interpreted as a measure of the amount of analyte in the sample.

35 How does the ELISA method work?

The antigen utilized in the ELISA method is bound to a solidphase.

Tubes and microplates made of rigid polystyrene, polyvinyland polypropylene are used as the solid phase. The microplatesused must be able to appropriately adsorb the antigen and the anti-body, but not adsorb the components in the other phases.106, 107

The enzymes that can be employed in ELISA include beta galac-tosidase, glucose oxidase, peroxidase, and alkaline phosphatase. Alkaline phosphatase can be stored at 4ƕC with its conjugate sodium azide. Alkaline phosphatase and P-nitro- phenyl phosphateare used as substrates are available in safe tablet forms, and produce a yellow color in positive reactions. For the peroxidase conjugate, 5 amino salicylic acid and orthophenylenediamine are used as the substrates and the production of a brown color is considered a positive reaction. If beta galactosidase is used, the sample must be read in a fluorometer. The catabolic effects ofenzymes determine both the acceleration and the specificity ofthe immunological reaction during the enzyme-substrate reaction.108

The enzyme-substrate reaction is usually completed within30–60 min.

The reaction can be stopped using sodium hydroxide (NaOH), hydrochloric acid

(HCl) or sulfuric acid (H2SO4).109The results are read on a spectrophotometer and at

400–600 nmdepending on the characteristics of the conjugate used.

36 Term110 Definition Solid phase Usually a microtiter plate well. Specially prepared ELlSA plates are commercially available. These have an 8×12- well format and can be used with a wide variety of specialized equipment designed for rapid manipulation of samples, including multichannel pipets. Adsorption The process of adding an antigen or antibody, diluted in buffer, so that itattaches passively to the solid phase on incubation. This is a simple way forimmobilization of one of the reactants in the ELISA and one of the mainreasons for its success. Washing The simple flooding and emptying of the wells with a buffered solution toseparate bound (reacted) from unbound (unreacted) reagents in the ELISA. Again, this is a key element to the successful exploitation of the ELISA. Antigens A protein or carbohydrate that when injected into animals elicit the production of antibodies. Such antibodies can react specifically with the antigen used and therefore can be used to detect that antigen. Antibodies Produced in response to antigenic stimuli. These are mainly protein in nature.In turn, antibodies are antigenic. Antispecies Produced when proteins (including antibodies) from one species are Antibodies injectedinto another species. Thus, -guinea pig serum injected into a rabbit elicits theproduction of rabbit anti-guinea pig antibodies. Enzyme A substance that can react at low concentration as a catalyst to promote aspecific reaction. Several specific enzymes are commonly used in ELISA with their specific substrates. Enzyme An enzyme that is attached irreversibly to a protein, usually an conjugate antibody. Thus, an example of antispecies enzyme conjugate is rabbit antiguinea linked to horseradish peroxidase. Substrate A chemical compound with which an enzyme reacts specifically. This reaction is used, in some way, to produce a signal that is read as a colour reaction (directly as a colour change of the substrate or indirectly by its effect on another chemical). Substrate A chemical that alters colour as a result of an enzyme interaction with substrate.

37 Stopping The process of stopping the action of an enzyme on a substrate. It has theeffect of stopping any further change in colour in the ELISA. Reading Measurement of colour produced in the ELISA. This is quantified using special spectrophotometers reading at specific wavelengths for the specific colours obtained with particular enzyme-chromophore systems. Tests can be assessed by eye.

Table:4 Terms and Definition

Generations111

¾ First generation assays are purified or unpurified native virus.

¾ Second generation assays use recombinant or synthetic viral peptide instead of viral lysate.

¾ Third generation assays are synthetic viral polypeptides are artificially produced by chemical synthesis.

¾ Fourth generation assays aresynthetic viral polypeptides are artificially produced by chemical synthesis, detectboth antigen and antibody in a single reaction chamber.

Types

Direct ELISA106, 112

Enzyme-labelled primary antibody is used, meaning that secondary antibodies are not needed. The enzyme-labelled primary antibody "directly" binds to the target (antigen) that is immobilized to the plate (solid surface). Next, the enzyme linked to the primary antibody reacts with its substrate to produce a visible signal that can be measured. In this way, the antigen of interest is detected.

Figure:12 Direct ELISA

INDIRECT ELISA113

In indirect ELISA, both a primary antibody and a secondary antibody are used. But in this case, the primary antibody is not labeled with an enzyme. Instead, the secondary antibody is labeled with an enzyme.

The primary antibody binds to the antigen immobilized to the plate, and then the enzyme-labeled secondary antibody binds to the primary antibody. Finally,

39 the enzyme linked to the secondary antibody reacts with its substrate to produce a visible signal that can be measured.

Figure:13 Indirect ELISA

Sandwich ELISA114, 115

In direct and indirect ELISA, it is the antigen that is immobilized to the plate. In sandwich ELISA, however, it is the antibody that is immobilized to the plate, and this antibody is called capture antibody. In addition to capture antibody, sandwich ELISA also involves the use of detection antibodies, which generally include the unlabeled primary detection antibody and the enzyme-labeled secondary detection antibody.

Firstly, the antigen of interest binds to the capture antibody immobilized to the plate. Secondly, the primary detection antibody binds to the antigen. Thirdly, the secondary detection antibody binds to the primary detection antibody, and then the enzyme reacts with its substrate to produce a visible signal that can be measured.

Figure:13 Sandwich ELISA

Competitive ELISA116

Compared with the three ELISA types above, competitive ELISA is relatively complex because it involves the use of inhibitor antigen, so competitive

ELISA is also known as inhibition ELISA. In fact, each of the three formats, direct, indirect, and sandwich, can be adapted to the competitive format. In competitive

ELISA, the inhibitor antigen and the antigen of interest compete for binding to the primary antibody. Here is a procedure of competitive ELISA:

Firstly, the unlabeled primary antibody is incubated with the sample containing the antigen of interest, leading to the formation of antigen-antibody complex (Ag-Ab). In this step, the antibody is excessive compared with the antigen, so there are free antibodies left.

Secondly, the Ag-Ab mixture is added to the plate coated with inhibitor antigen that can also bind to the primary antibody. The free primary antibody in the mixture binds to the inhibitor antigen on the plate, while the Ag-Ab complexes in the mixture do not and are therefore washed off.

41 Thirdly, the enzyme-labeled secondary antibody is added to the plate and binds to the primary antibody bound to the inhibitor antigen on the plate.

Finally, a substrate is added to react with the enzyme and emit a visible signal for detection.

Through this procedure, you may find that the final signal is inversely associated with the amount of the antigen of interest in the sample, meaning that the more antigen in the sample, the weaker the final signal. This is because primary antibodies bound to sample antigen will be washed off, while free primary antibodies left will be captured by inhibitor antigen immobilized to the plate and be measured by an enzymatic reaction.

Competitive ELISA

Figure:15 Competitive ELISA

42 Advantages Disadvantages Direct 1. Simple protocol, time- 1. High background. 110,117 ELISA saving, and reagents- 2. No signal amplification, since saving. only a primary antibody is used 2. No cross-reactivity from and a secondary antibody is not secondary antibody. needed. 3. Low flexibility, since the primary antibody must be labeled. Indirect 1. Signal amplification, since 1. Complex protocol compared ELISA110,117 one or more secondary with direct ELISA. antibodies can be used to 2. Cross-reactivity from bind to the primary secondary antibody. antibody. 2. High flexibility, since the same secondary antibody can be used for various primary antibodies. Sandwich 1. High flexibility. 1. The antigen of interest must be 110,117 ELISA 2. High sensitivity. large enough so that two different antibodies can bind to 3. High specificity, since different antibodies bind to it at different epitopes. the same antigen for 2. It's sometimes difficult to find detection. two different antibodies that recognize different epitopes on the antigen of interest and cooperate well in a sandwich format. Competitive 1. High flexibility. 1. Relatively complex protocol. 110,117 ELISA 2. High sensitivity. 2. Needs the use of inhibitor 3. Best for the detection of antigen. small antigens, even when they are present in low concentrations.

Table:5 Types of ELISA - Advantage and Disadvantage

43 Determination of Need and Requirement for Testing:118

CFR and AABB Standards both state that donors must be notified of any medically significant abnormality detected through testing. Additionally, regulatory and standard requirements involve tracing donations given before the positive unit

(Look back) and that recipients of donor’s previous donations must be notified and recommended to be tested in case donor was in window period when transfused unit was collected.

Approach to Donor Testing:

Early HIV testing:

The first generation EIA was developed for detection of AIDS in the year

1985, detected only the M-group specific HIV antigens not detect the Non M- subgroup and HIV-2 antigens. As this first generation assay used the cell lysate antigens had frequently nonspecific reactions between antibodies and antigens, the western blot analysis is a confirmatory method for the first generation tested samples. The first generation EIA was used for blood bank screening program.119,120

The second generation EIA was developed in the year 1987. In the second generation EIA, the HIV recombinant antigens and peptides was used instead of whole viral lysate bound in solid phase [The second generation assay was developed by recombinant DNA technology or synthetic peptide antigens M, N, O group].121,122

The Third generation EIA was developed in the year 1994.The third generation EIA was designed in a sandwich format. Recombinant HIV- 1 and HIV-2

44 proteins/peptides bound on the solid phase of the micro plate to react with patient serum.123

The fourth generation EIA was developed in the year 1997. Fourth generation EIA was developed to reduce the diagnostic window period. The fourth generation assay detects the HIV p24 antigen with anti HIV IgG and IgM antibodies.124

HIV p24 Antigen:125

HIV antigen (p24 antigen) testing was approved for testing blood donors in 1996. p24 testing was a small advancement; closing the window period to 16 days. The most promising method to further decrease the window period was a test that identified the presence of HIV nucleic acid.

HIV ELISA

Year Generation Detection Window period days 1985119 FIRST M –group specific HIV 1 antibody 6-12 weeks 1987121 SECOND M, N, O group antibody 33-35 days 1994123 THIRD HIV 1 & 2 antibody 22 days 1997124 FOURTH HIV 1 & 2 antibody, p24 antigen 16 days

Table:6 HIV ELISA -Generations

BridonM’baya et al.,126concluded that Gen screen ULTRA HIV antigen-antibody reagents (Bio-Rad, France) were utilised using the Evolis semi- automated platform. These were fourth generation enzyme immunoassays (EIA) that detect antibodies to HIV-I (groups M and O) and HIV-II as well as the HIV p24 antigen. The algorithm involved repeating in duplicate all initial positives and

45 interpreting results based on concordant two of the three. However, since 2015 a third category of discordant was added being those who test positive on EIA and negative on rapid test kit. HIV prevalence 1.9% in 2015 and has declining trend.

Repeat blood donors had a significantly lower prevalence for each TTI than first time blood donors.

Patel et al.,127reported that the presence of AHI in 23 specimens from 27 patients with AHI (85.1%) was detected and that the 4th-generation test detected 10 of the 13 cases of AHI that were missed by the 3rd-generation test. The 4 AHI cases missed by the 4th-generation test had low viral load (median of 6961 copies/mL).

Alagarraju et al.,128Compared the 4th-Generation HIV

Antigen/Antibody Combination Assay With 3rd-Generation HIV Antibody Assays for the Occurrence of False-Positive and False-Negative Results In this study, only 5 of the 24 specimens that harbored AHI and tested positive by NAAT (20.8%) could not be detected as such by the of 4th-Generation HIV assay. In contrast, the 3rd-

Generation HIV assay could not detect the presence of HIV in 10 of the specimens with positive results via NAAT (41.6%); the other 3rd-generation tests could not detect HIV in 16 of the specimens that had positive NAAT results (66.7%). These results confirm that the 4th-generation HIV assay is relatively more accurate than the 3rd-generation HIV assays in detecting acute HIV infection.

Bernard weber et al.,129 stated that the residual risk of HIV transmission by blood donation (mostly by viremic but antibody- negative donors) is 1/493,000 per unit in the United States. By additional screening for p24 Ag, the risk of HIV infection may be reduced to 1/676,000 per unit. The sensitivity and specificity of 3rd generation assay are 70% and 93.4% respectively with a predictive value(PPV-

46 72.1%, NPV-92.8%). The sensitivity and specificity of 4th generation assay are

88.2% and 97.2 % respectively with a predictive value(PPV 96.2%, NPV 96.2 %).

Kola Sujatha and KasiSeshuVaisakhi R etal.,130 concluded that 3rd

Generation V/S 4th Generation ELISA in Blood Donors for Early Diagnosis of HIV in Rural Population study shows that p24 Antigen detection by Fourth-Generation

ELISA is more sensitive for the detection of HIV infection, compared to Antibody detection by third-Generation ELISA and COOMBAIDS Dot Immuno Assay. This study emphasizes the importance of using Fourth-Generation assays, which permit the simultaneous detection of p24 antigen and antibodies to HIV-1 and 2, in the screening of all groups of persons for HIV infection, in order to ensure early detection of HIV infection by detecting p24 Ag, present in recent HIV infection prior to seroconversion.

Elizabeth O. Mitcell et al.,6 compared the analytical sensitivity of the two (2) 4thgeneration HIV immunoassays as well as the seroconversionsensi-tivity and specificity of the 4th and 3rd generation immunoassays,confirmed that the 4th generation HIV assays are superior in sen-sitivity to the 3rd generation assays, detecting HIV infection 7–11days earlier in the panels tested in this study, and detecting 94.9%(37/39) of the 39 HIV-1 RNA positive samples tested, compared to76.9% (30/39), detected by the 3rd generation assays.

Early HBV testing:

In earlier days infection with HBV was detected by demonstration of antibody titer by Complement Fixation Test. The first solid phase sandwich radio immunoassay named Ausria 125 was developed by Ling et al.

47 HBsAg:

Identified in an infected donors’ serum or plasma by enzyme immunoassays (EIA) using animal antibodies (anti-HBs) as the solid-phase capture reagent and conjugated anti-HBs as the probe. Chemiluminescent labels have replaced enzyme conjugates and chromogenic detection methods in automated testing.

S.no Generation Coated Window period 1. THIRD Monoclonal HBsAg antibody 37 days22 2. Monoclonal HBsAg antibody + 24 days22 FOURTH Polyclonal HBsAg antibody

Table:7 HBsAg ELISA-Generation

Anti-HBc:

Anti-HBc testing was implemented in the mid 1980s as a surrogate marker to reduce the transmission of other hepatidities. The tests utilized for donor screening detect both IgM and IgG. This frustrates donors who follow up with their physician. Typically only IgM is tested for in the clinical setting as the concern is current infection. Donors come back to blood centers believing that our results were wrong.

Cyrille Bisseye et al.,131 This study evaluated for the first time in blood donors in Libreville by the 4th generation ELISA (EVOLIS BIO-RAD) and electrochemiluminescence (Cobas 6000 e601) (using the Genscreen ULTRA HIV Ag-Ab,

MONOLISA HBsAg Ultra and Monolisa HCV Ag-Ab ULTRA (Bio-Rad, Marnes- la- Coquette, France) tests in the detection of HIV, HBV and HCV. The agreement

48 between the two tests was very good for the detection of HIV and HBsAg while it was fair for HCV detection. The use of reference molecular tests would make it possible to determine the sensitivity and specificity of these two tests in the detection of HIV, HBsAg and HCV in blood donors in Gabon.

Early Anti-HCV:

Figure:15 HCV ELISA generations

HCV ELISA

S.no Generation Coated Window Period 1. FIRST c100-3 epitope(NS4 region) 12-26 weeks 2. FIRST GEN + c22-3 and c33c 82 days SECOND (NS3 regions) 3. THIRD SECOND GEN + NS5 REGION 59 – 66 days22,134 4. FOURTH THIRD GEN + Core Antigen 10 – 21 days22,134

Table:8 HCV ELISA-Generations

49 Supplemental anti-HCV test and significance of signal-to-cut off (S/CO) ratio

Recombinant immunoblot assays (RIBA) were used in the past as supplemental assays to confirm serological reactivity by ELISA, but are now clinically obsolete with the availability of molecular tests.

François Rouet et al.,132 Usefulness of a Fourth Generation ELISA

Assay for the Reliable Identification of HCV Infection in HIV-Positive Adults from

Gabon (Central Africa) in their study Direct HCV assays are urgently required in

Resource Limited Settings. The HCV core Ag assay used in this study has many advantages: this test is not prone to sample carryover contamination, and is both easy to use and relatively cheap, in comparison with quantitative molecular HCV assays Nonetheless, they obtained five samples which were Ag-/RNA+, probably due to the 1:10 dilution that was performed prior the measurement of HCV core Ag concentrations. Thus, the sensitivity and specificity of the HCV core Ag assay for the determination of HCV status, as well as its utility for treatment monitoring, merit further evaluation in RLS.

Laperche et al.,134stated that the simultaneous detection of HCV antigen and antibodies provides a valuable alternative to methods that directly detect viremia, such as NAT or an HCV Ag-specific assay, especially with regards to cost, organization, emergency, and logistic difficulties. It could be implemented for blood screening in developing countries where NAT or HCV Ag assays are not affordable or not feasible.

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y w j r 6 M 9 n C 2 F M B a 1 1 2 3 3 e 3 3 1 1 1 J HIV HIV HBsAg HBsAg HCV HBsAg HCV HCV t

i 3rd Gen 70% 76.90% 99.30% y s t i n v e i

S 4th gen 88.20% 94.90% 100% 100% 100% 99.10% 100%

i 3rd Gen 93.40% 93.40% 99.70% y c t e i c p i

S 4th gen 970%.2 99.70% 100% 100% 100% 99.90% 100%

3rd Gen 72.10% 72.10% V

P 4th gen 96.40% 99.37% 100% 100% 100% 100%

3rd Gen 92.80% 92.80% V

N 4th gen 96.40% 100% 100% 100% 100%

A fourth-generation antigen and antibody assay (combination EIA) is more convenient as two infectious markers for HIV and HCV are detected in the same assay. fourth-generation HBsAg assay is more sensitive when compared to third-generation. In resource constrained countries TTI screening is highly variable, depending upon infrastructure, trained manpower and financial resource.

Implementation of fourth-generation ELISA will result in dramatic reductions of

TTI and relative risk. However, minimal residual risk still remain.

MATERIALS AND

METHODS

MATERIALS AND METHODS

STUDY METHOD

This Cross-sectional study was conducted over one year period from August 2018- July 2019 in the Department of Transfusion Medicine, The Tamil Nadu Dr.MGR Medical University, Guindy, Chennai. A total of 373 voluntary blood donors wereselected.

The study was approved by the ethical committee of The Tamil Nadu Dr. MGR Medical University, Chennai. The donors were classified as upper, middle and lower socioeconomic status based on Kuppusamyclassification.39

SAMPLE COLLECTION

Serum was separated from donated samples of voluntary blood donors in a sterile plain test tube and stored at -20oC for ELISA and PCR tests.

INCLUSION CRITERIA

x Those voluntary blood donors who fulfil the criteria as per DGHS guidelines.

Those blood donors who are willing to participate in thestudy.

EXCLUSION CRITERIA

x Those voluntary blood donors who do not fulfill the donor selection criteria as per the DGHSguidelines.

x Family and Replacement donors are not included in this study.

x Those blood donors who are not willing to participate in thestudy

52 ESTIMATED SAMPLE SIZE

ZĮ2PQ x L2

x =Į- Level of significance = 5%

x P - Prevalence = 1.03%

x Q - 100-P = 98.97%

x L2 – Allowable Error = 3%

25 x 1.03 x 98.97 x 284 9

x Minimum required sample size = 284

STUDY PERIOD

The total sample size was split month wise from August 2018 to July 2019.

STATISTICAL ANALYSIS

x Data analysis was done using SPSSsoftware

x Demographic details were given in descriptivestatistics

x Quantitative data was given in summarystatistics

x P<0.05 was consideredsignificant

METHOD OFSCREENING

The samples that were frozen earlier were thawed and used. All blood units were tested for HIV I&II, HCV antibodies and HBsAg using 3rd generation

ELISA (Merilisa hiv 1 & 2 gen 3,28 Merilisa HCV29 and Merlisa HBsAg30 kits manufactured by Meril diagnostics). In addition donor units were screening with 4th

53 generation HIV Ag-Ab, HCV Ag-Ab and HBsAg ELISA (Genscreen ULTRA HIV

Ag-Ab kits,32 monolisa HCV Ag-Ab ULTRA V2 kit33 and monolisa HBsAg ULTRA

ELISA kits34 manufactured by Bio Rad).

3rd generation ELISA 4th generation ELISA VIRUS Detection Detection Ab GP120 /GP41, Ab GP120 /GP41, HIV Ab GP36 Ab GP36, P24 Ag. Anti HCV (NS1,NS2,NS3,NS4 & Anti HCV (NS1,NS2,NS3,NS4 & HCV NS5) NS5) Capsid Ag HBsAg & 11 subtypes HBV HBsAg most part of variants HBV strains

Table:9 3rd and 4th generation ELISA of HIV, HCV and HBsAg.

Manufacturer’s instructions were strictly followed while performing each assay. Samples with absorbance values less than 10% of the cut-off value were considered to be ELISA non-reactive.

Sample with absorbance values equal to greater than the cut-off value were initially considered to be ELISA reactive. Sample with absorbance values within 10% of cut off value were considered in grey zone.

Samples initially found in grey zone were retested using the same ELISA kit and if again found in grey zone were termed as possibly reactive and included in analysis.

The seroreactive samples will be confirmed by RT-PCR.

54 ASSAY PROCEDURE

3RD GENERATION ANTI-HIV I&II (Merilisa hiv 1 & 2 gen 3)28

1. All reagents were brought to the room temperature (20-250C) before starting thetest.

2. All reagents were mixed gently prior to use without inducingfoaming.

3. A clean, disposable tip should be used for dispensing each control and sample.

4. Add 50ȝl sample diluent to A-1 well as blank.

5. Add 50ȝl Negative Control in each well no. B1 to D1 and 50ȝl of the anti HIV Control into wells E-1 & F-1 respectively.

6. Add 50ȝl sample from G-1 onwards .

7. Cover the wells with adhesive strip(s) and incubate for 30 mins at +37ºC ± 1ºC

8. At the end of the incubation, discard the content of the plate blot to the plate on absorbent paper.

9. Fill the wells with wash buffer(350ȝl) and allow to soak for 30 seconds thereafter decant the buffer and blot the plate on absorbent paper. Repeat the steps for 4 additional times. (Total 5 washes)

10. Immediately after washing the plate Add 100 ȝl of Conjugate to all wells except blank(A1).

11. Cover the wells with adhesive strip(s) and incubate for 30 minutes at +37ºC ± 1ºC

55 12. At the end of the incubation time wash the plate as described in steps 8 and 9.

13. Add 100 ȝl of substrate solution to each well.

14. Cover the wells with adhesive strip(s) and incubate Incubate at room temperature (20 - 30ºC) for 30 minutes in dark.

15. Add 100 ȝl of stop solution to each well.

16. Within 15 minutes read absorbance at 450 nm using 630 nm as the reference wavelength

MICROPLATE

1 2 3 4 5 6 7 8 9 10 11 12 A BL B NC C NC D NC E PC F PC G S1 H S2 BL-Blank, NC-Negative Control, PC-Positive Control, S-Sample

CALCULATION OFRESULTS:

Negative Control

Calculation of mean absorbance of the Negative Control

Mean Negative Control(NCਅ) = (Abs B1+ Abs C1+ Abs D1) / 3

56 Cut-Off Value Calculation

Cut-off value = NCਅ + 0.2

ASSAY PROCEDURE

3RD GENERATION ANTI-HCV (Merilisa HCV)29

1. All reagents were brought to the room temperature (20-250C) before starting thetest.

2. All reagents were mixed gently prior to use without inducingfoaming.

3. A clean, disposable tip should be used for dispensing each control and sample.

4. Add 100ȝl sample diluent to A-1 well as blank.

5. Add 10ȝl Negative Control in each well no. B1 to D1 and 10ȝl of the anti HCV Control into wells E-1 & F-1 respectively.

6. Add 10ȝl sample from G-1 onwards .

7. Cover the wells with adhesive strip(s) and incubate for 30 minutes at +37ºC ± 1ºC

8. At the end of the incubation, discard the content of the plate blot to the plate on absorbent paper.

57 10. Immediately after washing the plate Add 100 ȝl of Conjugate to all wells except blank(A1).

11. Cover the wells with adhesive strip(s) and incubate for 30 mins at +37ºC ± 1ºC

12. At the end of the incubation time wash the plate as described in steps 8 and 9.

13. Add 100 ȝl of substrate solution to each well.

14. Cover the wells with adhesive strip(s) and incubate Incubate at room temperature (20 - 30ºC) for 30 minutes in dark.

15. Add 100 ȝl of stop solution to each well.

16. Within 15 minutes read absorbance at 450 nm using 630 nm as the reference wavelength.

MICROPLATE

1 2 3 4 5 6 7 8 9 10 11 12 A BL B NC C NC D NC E PC F PC G S1 H S2 BL-Blank, NC-Negative Control, PC-Positive Control, S-Sample

58 CALCULATION OFRESULTS:

Negative Control

Calculation of mean absorbance of the Negative Control

Mean Negative Control(NCਅ) = (Abs B1+ Abs C1+ Abs D1) / 3

Cut-Off Value Calculation

Cut-off value = NCਅ + 0.2

ASSAY PROCEDURE

3RD GENERATION HBsAg (HBsAg)30

1. All reagents were brought to the room temperature (20-250C) before starting thetest.

2. All reagents were mixed gently prior to use without inducingfoaming.

3. A clean, disposable tip should be used for dispensing each control and sample.

4. 25ȝl of conjugate 1 (R6) in each well

5. 75ȝl of HIV Ag positive control (R5) in well A1, 75ȝl of HIV Ab positive control (R4) in well B1 and 75ȝl of negative control (R3) in well C1, D1 and E175 ȝl of specimen 1 in well F1

1. 75 ȝl of specimen 2 in well G1, etc.

2. Add 50ȝl Negative Control in each well no. B1 to D1 and 50ȝl of the Positive Control into wells E-1 & F-1 respectively.

59 3. Add 50ȝl sample from G-1 onwards. Keep the first well as blank which is optional(A1).

4. Add 100 ȝl of Conjugate to all wells except blank(A1).

5. Cover the wells with adhesive strip(s) and incubate for 60 minutes at +37ºC ± 1ºC

6. At the end of the incubation, discard the content of the plate blot to the plate on absorbent paper.

7. Fill the wells with wash buffer(350ȝl) and allow to soak for 30 seconds thereafter decant the buffer and blot the plate on absorbent paper. Repeat the steps for 4 additional times. (Total 5 washes)

8. Cover the wells with adhesive strip(s) and incubate Incubate at room temperature (20 - 30ºC) for 30 minutes in dark.

9. Add 100 ȝl of stop solution to each well.

10. Within 15 minutes read absorbance at 450 nm using 630 nm as the reference wavelength

MICROPLATE

1 2 3 4 5 6 7 8 9 10 11 12 A BL B NC C NC D NC E PC F PC G S1 H S2 BL-Blank, NC-Negative Control, PC-Positive Control, S-Sample

60 CALCULATION OFRESULTS:

Negative Control

Calculation of mean absorbance of the Negative Control

Mean Negative Control(NCਅ) = (Abs B1+ Abs C1+ Abs D1) / 3

Cut-Off Value Calculation

Cut-off value = NCਅ + 0.1

ASSAY PROCEDURE

HIV ELISA 4TH GENERATION ( Gencreen ULTRA HIV Ag-Ab)32

1. All reagents were brought to the room temperature (20-250C) before starting thetest.

2. All reagents were mixed gently prior to use without inducingfoaming.

3. A clean, disposable tip should be used for dispensing each control and sample.

4. 25ȝl of conjugate 1 (R6) in each well

5. 75ȝl of HIV Ag positive control (R5) in well A1, 75ȝl of HIV Ab positive control (R4) in well B1 and 75ȝl of negative control (R3) in well C1, D1 and E1

6. 75ȝl of sample from F1 onwards.

7. Cover the wells with adhesive strip(s) and incubate the microplate at +37ºC ± 1ºC for 1 hour ± 4 minutes.

8. At the end of the incubation, discard the content of the plate blot to the plate on absorbent paper.

61 9. Fill the wells with wash buffer(370ȝl) and allow to soak for 30 seconds thereafter decant the buffer and blot the plate on absorbent paper. Repeat the steps for 2 additional times. (Total 3 washes).

10. Quickly dispense 100 ȝl of conjugate 2 solution (R7a + R7b) into all wells, the conjugate must be shaken before use.

11. Cover the wells with adhesive strip(s) and incubate for 30 minutes (± 4 minutes) at room temperature(18 - 30ºC).

12. At the end of the incubation time wash the plate as described in steps 8 and 9.

13. Quickly dispense into each well 80ȝl of prepared enzymatic developed solution (R8+R9) in all wells. Allow the reaction to develop in the dark for 30 (± 4 minutes) minutes at room temperature (18 - 30°C).

14. Add 100 ȝl stopping solution (R10) in all wells

15. Within 30 minutes read optical density at 450 / 620-700 nm using a plate reader

MICROPLATE

1 2 3 4 5 6 7 8 9 10 11 12 A PC-Ag B PC-Ab C NC D NC E NC F S1 G S2 H S3 PC-Ag – HIV Ag Positive Control, PC-Ab – HIV Ab Positive Control, NC-Negative Control, S-Sample.

62 CALCULATION OFRESULTS:

Negative Control

Calculation of mean absorbance of the Negative Control

Mean Negative Control(NCਅ) = (OD C1+ OD D1+ OD E1) / 3

Cut-Off Value Calculation

Cut-off value = NCਅ + 0.2

ASSAY PROCEDURE

HCV ELISA 4TH GENERATION ( Monolisa HCV Ag-Ab ULTRA V2)33

1. All reagents were brought to the room temperature (20-250C) before starting thetest.

2. All reagents were mixed gently prior to use without inducingfoaming.

3. A clean, disposable tip should be used for dispensing each control and sample.

4. 100ȝl of conjugate 1 (R6) in each well

5. 50ȝl of negative control (R3) in well A1, 50ȝl positive control (R4) in well B1, C1, D1 and working positive control Ag(R5a + R5b)in well E1

6. 50ȝl of sample from F1 onwards.

7. Cover the wells with adhesive strip(s) and incubate the microplate for 90 ± 5 minutes at 37ºC ± 1ºC.

8. At the end of the incubation, discard the content of the plate blot to the plate on absorbent paper.

63 9. Fill the wells with wash buffer(370ȝl) and allow to soak for 30 seconds thereafter decant the buffer and blot the plate on absorbent paper. Repeat the steps for 4 additional times. (Total 5 washes).

10. Quickly dispense 100 ȝl of conjugate 2 solution (R7) into all wells, the conjugate must be shaken before use.

11. Cover the wells with adhesive strip(s) and incubate for 30 minutes (± 5 minutes) at 37ºC ± 1ºC.

12. At the end of the incubation time wash the plate as described in steps 8 and 9.

13. Quickly dispense into each well 80ȝl of prepared enzymatic developed solution (R8+R9) in all wells.Allow the reaction to develop in the dark for 30 (± 5 minutes) minutes at room temperature (18 - 30°C).

14. Add 100 ȝl stopping solution (R10) in all wells

15. Within 30 minutes read optical density at 450 / 620-700 nm using a plate reader

MICROPLATE

1 2 3 4 5 6 7 8 9 10 11 12 A NC B PC C PC D PC E PC-Ag F S1 G S2 H S3 PC-Ag – HCV Ag Positive Control, PC- Positive Control, NC-Negative Control, S- Sample.

64 CALCULATION OFRESULTS:

Negative Control

Calculation of mean absorbance of the Negative Control

Mean positive control (PCਅ) = (OD B1+ OD C1+ OD D1) / 3

Cut-Off Value Calculation

Cut-off value = Mean OD PC /5

ASSAY PROCEDURE

HBsAg ELISA 4TH GENERATION ( Monolisa HBsAg ULTRA)34

1. All reagents were brought to the room temperature (20-250C) before starting thetest.

2. All reagents were mixed gently prior to use without inducingfoaming.

3. A clean, disposable tip should be used for dispensing each control and sample.

4. 100 ȝl of negative control (R3) in well A1,B1,C1 and D1.

5. 100ȝl positive control (R4) in well E1.

6. 100ȝl of sample from F1 onwards.

7. Quickly dispense 50 ȝl of conjugate solution (R6 + R7) into all wells

8. Cover the wells with adhesive strip(s) and incubate the microplate for 90 ± 5 minutes at 37ºC ± 1ºC.

65 9. At the end of the incubation, discard the content of the plate blot to the plate on absorbent paper.

10. Fill the wells with wash buffer(370ȝl) and allow to soak for 30 seconds thereafter decant the buffer and blot the plate on absorbent paper. Repeat the steps for 4 additional times. (Total 5 washes).

11. Quickly dispense into each well 100ȝl of prepared enzymatic developed solution (R8+R9) in all wells.Allow the reaction to develop in the dark for 30 (± 5 minutes) minutes at room temperature (18 - 30°C).

12. Add 100 ȝl stopping solution (R10) in all wells

13. Within 30 minutes read optical density at 450 / 620-700 nm using a plate reader

MICROPLATE

1 2 3 4 5 6 7 8 9 10 11 12 A NC B NC C NC D NC E PC F S1 G S2 H S3 PC- Positive Control, NC-Negative Control, S-Sample.

66 CALCULATION OFRESULTS:

Negative Control

Calculation of mean absorbance of the Negative Control

Mean Negative control (NCਅ) = ( OD A1+OD B1+ OD C1+ OD D1) / 4

Cut-Off Value Calculation

Cut-off value = NCܫ + 0.050.

REAL TIME –PCR

PRINCIPLE – Real time PCR:

Pathogen detection by the polymerase chain reaction (PCR) is based on the amplification of specific regions of the pathogen genome. In real-time PCR the amplified product is detected via fluorescent dyes. These are usually linked to

Oligonucleotide probes that bind specifically to the amplified product. Monitoring the fluorescence intensities during the PCR run (i.e., in real time) allows the detection and quantitation of the accumulating product without running in agarose gel electrophoresis following by gel documentation.

DETECTION:

Real-time PCR Kit constitutes a ready to- use system for the detection of viruses using polymerase chain reaction (PCR). It contains reagents and enzymes for the specific amplification of 145bp region of the HBV/HCV/HIV genome, and for the direct detection of the specific amplicon in fluorescence channels Cycling Green.

In addition, it contains an Internal control amplification system to identify possible

PCR inhibition. External positive controls are supplied, which can be used as both qualitative and quantitative to determination the amount of viral load.

67 PRECAUTIONS:

• Store positive material (Specimens, Standards or amplicons) separately from all other reagents and add it to the reaction mix in a separate facility.

• All the positive standards & specimens should be mixed & dispensed in extraction area.

• All the reagents including the NTC (except for standards & specimens) should be mixed & dispensed in pre-mix area.

• Use pipette tips with filters only.

• Always use disposable powder-free gloves.

MATERIALS REQUIRED:

• 0.2 ml PCR tubes / 8 well strips / 96 well plate according to real

time PCR machine & Model.

• Micro Pipettes Variable Volume 0.5-10ȝl, 10-100ȝl, and 100-

1000ȝl

• Sterile pipette tips with aerosol barrier 2-20ȝl, 10-100ȝl, and 100-

1000ȝl

• Disposable powder-free gloves

• Centrifuge with 1,5ml tubes rotor

• 1.5ml/2ml centrifuge tubes

68 DETECTION PROTOCOL

Things to do before starting

Before each use, all reagents need to be thawed completely, mixed by

gently inverting and centrifuged briefly.

Make sure that Positive and Negative control is included in every PCR

run.

Make sure that internal control template is added during DNA

purification.

Detection Mix

HBV HCV HIV (HELINI)36 (HELINI)38 (HELINI)35 Components Volume Volume Volume Probe PCR Master Mix ȝl ȝl ȝl RT Enzyme - ȝl ȝl Taq Enzyme mix ȝl ȝl ȝl Primer Probe Mix HBV Primer HCV Primer HIV Primer Probe 2.5ȝl Probe2.5ȝl Probe2.5ȝl IC Primer Probe Mix 2.5ȝl 2.5ȝl 2.5ȝl [IC PP Mix] Purified DNA sample 10ȝl 10ȝl 10ȝl Total reaction volume 25ȝl 25ȝl 25ȝl

Table:10 RT-PCR Technique for HBV, HCV and HIV.

69 Negative Control setup [NTC]

Add 10ȝl of nuclease free water.

Qualitative Positive Control setup

Add 10ȝl of any one of the Positive controls [From QS1 to QS4]

Quantitative Positive controls setup

Include all Positive controls prepared from QS1 to QS4.

Amplification Protocol

Steps Time Temperature Reverse Transcription (for 30 min 50ºC HCV & HIV) Taq enzyme activation 15min 95ºC Denaturation 20sec 95ºC 45 cycles Annealing/Data collection* 20sec 56ºC Extension 20sec 72ºC

Table:11 RT-PCR Amplification Protocol for HBV, HCV and HIV

HBV = FAM channel

Internal Control = HEX Channel

INTERPRETATION OF RESULTS:

Calculate copies per ml using following formula:

Result copies /PP l x Elution Volume l Result copies / ml Sample Volume ml

70 Calculate IU per ml using following formula:

Result IU /PP l x Elution Volume l Result IU / ml Sample Volume ml

Negative control

If a false positive occurs with one or more of the NTC reactions, sample contamination may have occurred. Invalidate the run and repeat the assay with stricter adherence to the procedure guidelines.

Positive Control

Positive control reactions should produce positive results before 36 cycles. If expected positive reactivity is not achieved, invalidate the run and repeat the assay with stricter adherence to the procedure guidelines.

Test Sample/Specimen - Positive

When all controls meet stated requirements, a test sample/specimen is considered presumptive positive.

Test sample/Specimen - Negative

When all controls meet the stated requirements, sample is considered negative.

RESULTS

RESULT

Thiscrosssectionalstudywasconductedon373voluntaryblooddonordona ted samples in Chennai, to compare the sensitivity of 3rd and 4th generation

ELISA kits and also to know the prevalence of HIV, HCV and HBsAg.

Table:12Gender distribution of the study group

S.no Sex Percentage % 1 Male 85.8 2 Female 14.2 Total 100.0

53Total No of voluntary donors: 373

320 Male Female

Figure :16 Gender distribution

Demographic analysis showed, of the 373 donors, 320 (85.8%) were males and 53 (14.2%) were females.

72 Age distribution among the 373 blood donors were 49.1% in 18-20 years, 32.4% in 21-30 years, 9.4% in 31-40 years, 6.7 % in 41-50 years, 2.4% in >50

Years. (Table ; Figure )

Table:13 Age distribution of the study group

S.no Age group In years Percentage % 1 18-20 49.1 2 21-30 32.4 3 31-40 9.4 4 41-50 6.7 5 >50 2.4 Total 100.0

Total No of voluntary donors: 373

NO OF DONOR

183 121

35 25 9 18-20 21-30 31-40 41-50 >50

Figure: 17 Age Distribution in years

Percentage distribution of blood donors on the basis of Occupation were

33.3% of working (employed), 64.3% of students, 2.4% of others.

Table:14 Distribution on the basis of occupation

S.no Occupation Percentage % 1 Working (employed) 33.3% 2 Students 64.3% 3 Others 2.4% Total 100

Total No of voluntary donors: 373

No of Donors

240

250

200 124 150

100 9 50

0 working(emplo Student others yed) No of Donors 124 240 9

Figure:18 Donor Occupation

Percentage distribution of blood donors on the basis of Occupation were

33.3% of working (employed), 64.3% of students, 2.4% of others.

Table :15 Distribution on the basis of socioeconomic status

S.no Socioeconomic status Percentage % 1 Upper 5.1 2 Middle 89.8 3 Lower 5.1 TOTAL 100.0%

Total No of voluntary donors: 373

No of Donors

350 335 300 250 200 150 100 50 19 0 19 High Middle Low

Figure:19 Socioeconomic status

Most of our donors belong to middle socioeconomic status (89.8%) followed by low (5.1%) and high (5.1%).(Table ; Figure ).

75 Zonal Distribution of Voluntary Blood Donors

90 83

70 62

s r o

n 50 o d

f 37 o 40 34 o

N 29 30 20 21 20 20 15 15 14

6 7 10 5 4 1 0 0 2 4 6 8 10 12 14 16 18 zone

Figure:20 Zone of Distribution

Total No of voluntary donors: 373

Zonal distribution of donors were on the basis of their residence in and around Chennai. Those within the city were divided further in to 15 zones. 290 donors were from 15 zones within Chennai city and 83 were from outside the city limits.

According to zones in the city there were 4% in zone 1, .3% in zone

2,1.3%inzone3,4%inzone4,1,1%inzone5,1.6%inzone6,9.1%in zone7,7.8%inzone8,1.9%inzone9,5.4%inzone10,9.9%inzone11, 5.6% in zone 12,

5.4% in zone 13, 16.6% in zone 14 and 3.8% in zone 15. The remaining 22.3% were from outside the limits of Chennai city.

76 Table:16 Distribution of Blood Group

S.no Blood group Percentage%

1 A1 22.8 2 B 27.9 3 O 41.3

4 A1B 7.2

5 A2B 0.5

6 A2 0.3 Total 100.0

Total No of voluntary donors: 373

Blood group of Donors

200 154 104 85 100 27 2 1 0 A1 B O A1B A2B A2 No of Donors 85 104 154 27 2 1

Figure:21 Distribution of Blood Group

Blood group distributions among the blood donors were 22.8% of ‘A’ group, 27.9% of ‘B’ group, 41.3% of ‘O’ group, 72% of ‘AB’ group, 0.5% of ‘A2B’ group and 0.3% of ‘A2’ group.

77 Table:17 Distribution of Rh type

S.no Rh Type Percentage % 1 Positive 94.1 2 Negative 5.9 Total 100.0

Total No of voluntary donors: 373

Positive Negative 351

Figure: 22 Rh Distribution

Rh distribution among donors were 351(94.1%) of Rh D positive group,

22(5.9%)of Rh negative group.

78 Table:18 Month wise Sample Distribution

Month No. of Samples in percentage Aug-18 9.4 Sep-18 7.8 Oct-18 11.0 Nov-18 8.0 Dec-18 2.7 Jan-19 8.8 Feb-19 10.7 Mar-19 8.6 Apr-19 7.8 May-19 7.8 Jun-19 8.3 Jul-19 9.1

Total No of voluntary donors: 373

No of sample

45 41 40 40 35 33 34 32 31 35 29 30 29 29 30 25 20 15 10 10 5 0

Figure:23 Month wise Sample Distribution

Table:19 HIV Screening by 3rd and 4th Generation ELISA

HIV Screening Reactive Nonreactive Indeterminate 3rd Generation 0 373 0 4th Generation 0 373 0

100 100 100

40 0 0 4th Generation 20 0 0 3rd Generation 0 Reactive Nonreactive Indeterminate

3rd Generation 4th Generation

Figure:24 HIV Screening by 3rd and 4th Generation ELISA

In our study there was none of the donor reactive for HIV by screening with 3rd and 4th generation HIV ELISA.

Table: 20 HCV Screening by 3rd and 4th Generation ELISA

HCV Screening Reactive Nonreactive Indeterminate 3rd Generation 0 373 0 4th Generation 1 372 0

99.74% 100% 100.00%

80.00%

60.00% 0.26% 40.00% 0 4th Generation 20.00% 0.00% 0.00% 3rd Generation 0.00% Reactive Nonreactiv Indetermin e ate 3rd Generation 0.00% 100% 0.00% 4th Generation 0.26% 99.74% 0

3rd Generation 4th Generation

Figure:25 HCV Screening by 3rd and 4th Generation ELISA

Among 373 donors, none of the donation seroreactive for HCV by screened with 3rd generation HCV ELISA whereas in 4th generation HCV Ag-Ab

ELISA 1 donation (0.26%) showed seroreactive.

Table: 21HBsAg Screening by 3rd and 4th Generation ELISA

HBsAg Screening Reactive Nonreactive Indeterminate 3rd Generation 3 370 0 4th Generation 3 370 0

3rd Generation 4th Generation % 0 2 . % 9 0 9 2 . 9 9 % 0 % 8 0 . 0 0 . % 0 0 8 % . 0 0

0 4th Generation . 0

REACTIVE 3rd Generation NONREACTIVE INDETERMINATE

Figure:26 HBsAg Screening by 3rd and 4th Generation ELISA

Among 373 donors, there was 3(0.80%) donation seroreactive for HBsAg by screening with 3rd generation HBsAg and 4th generation HBsAg-ULTRA ELISA.

82 Table: 22 Age distribution of seropositive of HCV & HBsAg BY

3rdGeneration ELISA

3rd generation 4th generation Age HCV HBsAg Total HCV HBsAg Total group In seroreactive seroreactive seroreactive seroreactive years (Total (Total Seroreactive (Total (Total Seroreactive donors) donors) donors) donors) 18-20 0(183) 0(183) 0(183) 0(183) 0(183) 0(183) 21-30 0(121) 3(121) 3(121) 1(121) 3(121) 4(121) 31-40 0(35) 0(35) 0(35) 0(35) 0(35) 0(35) 41-50 0(25) 0(25) 0(25) 0(25) 0(25) 0(25) >50 0(9) 0(9) 0(9) 0(9) 0(9) 0(9)

18-20 21-30 31-40 41-50 >50 0.035 3.30% 0.03 2.47% 2.47% 2.47% 0.025 0.02 0.015 0.83% 0.01 0.005 0 0 seroreactive - seroreactive Total seroreactive - seroreactive Total HCV HBsAg Seroreactive HCV HBsAg Seroreactive 3rd generation 4th generation

Figure:27 Age distribution of seropositive of HCV & HBsAg BY 3rdand 4thGeneration ELISA

Age distribution of seropositivity of the present study among the voluntary blood donors is seen in 21-30 years of age in which 3(121) donation

(2.47%) seroreactive for HBsAg by screening with 3rd generation HBsAg and 4th generation HBsAg-ULTRA ELISA. None of the donation seroreactive for HCV by screened with 3rd generation HCV ELISA whereas in 4th generation HCV Ag-Ab

ELISA 1(121) donation (0.83%) showed seroreactive.

83 Table: 23 Gender distribution of seropositive of HCV & HBsAg by 3rd &

4thGeneration ELISA

3rd generation 4th generation HCV HBsAg Total HCV HBsAg Total sex seroreactive seroreactive seroreactive seroreactive (Total (Total Seroreactive (Total (Total Seroreactive donors) donors) donors) donors) Male 0(320) 3(320) 3(320) 1(320) 3(320) 4(320) Female 0(53) 0(53) 0(53) 0(53) 0(53) 0(53) Total 3(373) 4(373)

0.014 1.25% 0.012 0.94% 0.94% 0.94% 0.01 0.008 0.006 0.31% 0.004 0.002 0 0 0 0 0 0 0 0

- - E E

E E E E V V V V I I I I V V T T I I

T T G G T T C C L L C C C C A A A A A A V V A A S S A E A E T T C C E E B B E E R R O O R R H H R R H H O T O T O O O O R R R R R E R E E E S S E E S S S S 3RD GENERATION 4TH GENERATION

Male Female

Figure:28 Gender distribution of seropositive of HCV & HBsAg by 3rd & 4thGeneration ELISA

Gender distribution of seropositive of present study, 3rd generation

ELISA showed 3(0.94%) out of 4 positive donations and did not pick up

1(0.31%) out of 4 HCV positive donation whereas 4th generation ELISA picked all 4 (1.25%) out of 4 as positive reactions (3 HBsAg and 1HCV). None of the female donors were seropositive.

84 Table : 24 Occupation distribution of seropositive of HCV & HBsAg by 3rd & 4thGeneration ELISA

3rd generation 4th generation HCV HBsAg Total HCV HBsAg Total Occupation seroreactive seroreactive seroreactive seroreactive (Total (Total Seroreactive (Total (Total Seroreactive donors) donors) donors) donors) Working 0(124) 2(124) 2(124) 1(124) 2(124) 03(124) (employed) Students 0(240) 1(240) 1(240) 0(240) 1(240) 1(240) Others 0(9) 0(9) 0(9) 0(9) 0(9) 0(9) Total 3(373) 4(373)

2.42% 0.025 0.02 1.61% 1.61% 1.61% 0.015 0.80% 0.01 0.42% 0.42% 0.42% 0.42% 0.005 0 0 0 0 0 0 0 0 0

- - E E

E E E E V V V V I I V I V I T T I I

T T G G T T C C L L C C C C A A A A A A V V A A S S A E A E T T C C E E B B E E R R O O H R H R R R H H O O T T O O O O R R R R R E R E E E S S E E S S S S 3RD GENERATION 4TH GENERATION

Working (employed) Students Others

Figure:29 Occupation distribution of seropositive of HCV & HBsAg by 3rd & 4thGeneration ELISA

In our study the prevalence of HBsAg among student sub group was

1(240) seropositive donation (0.42%) whereas working (employed) sub group was

2(133) seropositive donation (1.61%) in both 3rd and 4th generation HBsAg ELISA.

In HCV 1(133) seropositive donation (0.80%) was seen in working (employed) group by 4th generation ELISA which is not picked up by 3rd generation ELISA.

85 Table: 25 Socioeconomic status distribution of seropositive of HCV &

HBsAg by 3rd & 4thGeneration ELISA

3rd generation 4th generation HCV HBsAg HCV HBsAg Socioeconomic seroreactive status seroreactive seroreactive seroreactive (Total (Total donors) (Total donors) (Total donors) donors) High 0(19) 0(19) 0(19) 0(19) Middle 0(335) 3(335) 1(335) 3(335) Lower 0(19) 0(19) 0(19) 0(19) TOTAL 0(373) 3(373) 1(373) 3(373)

0.012 1.20% 0.01 0.90% 0.90% 0.90% 0.008 0.006 0.004 0.30%

0.002 0 0 0 0 0 0 0 0 0 0 0

- - E E

E E E E V V V V I I I I V V T T I I

T T T G T G C C L L C C C C A A A A A A V V A A S S A E A E T T C C E E B B E E R R O O R R H H R R H H O T O T O O O O R R R R R E R E E E S S E E S S S High Middle LowerS 3RD GENERATION 4TH GENERATION

Figure:30 Socioeconomic status distribution of seropositive of HCV & HBsAg BY 3rd & 4thGeneration ELISA

Socioeconomic status of seropositivity of the present study among the voluntary blood donors is seen in middle Socioeconomic status in which 3(335) donation (0.90%) seroreactive for HBsAg by screening with 3rd generation HBsAg and 4th generation HBsAg-ULTRA ELISA. None of the donation seroreactive for

HCV by screened with 3rd generation HCV ELISA whereas in 4th generation HCV

Ag-Ab ELISA 1(335) donation (0.30%) showed seroreactive.

86 Table: 26 Blood group distribution of seropositive of HIV, HCV & HBsAg by

3rd & 4thGeneration ELISA

3rd generation 4th generation HCV HBsAg HCV HBsAg Blood group seroreactive seroreactive seroreactive (Total seroreactive (Total (Total (Total donors) donors) donors) donors) A1 0(85) 2(85) 0(85) 2(85) B 0(104) 0(104) 0(104) 0(104) O 0(154) 1(154) 1(154) 1(154) A1B 0(27) 0(27) 0(27) 0(27) A2B 0(2) 0(2) 0(2) 0(2) A2 0(1) 0(1) 0(1) 0(1) TOTAL 0(373) 3(373) 1(373) 3(373)

A1 B O A1B A2B A2

0.025 2.35% 2.35% 2.35% 2.35%

0.02 1.30% 0.015 0.65% 0.01 0.65% 0.65% 0.65% 0.005 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 seroreactive - seroreactive Total seroreactive - seroreactive Total HCV HBsAg Seroreactive HCV HBsAg Seroreactive 3rd generation 4th generation

Figure:31 Blood group distribution of seropositive of HIV, HCV & HBsAg by 3rd & 4thgeneration ELISA

In 3rd generation ELISA identified 1 HBsAg positive belonged to blood group O whereas 4th generation ELISA picked 2 (1 HBsAg and 1 HCV) belonged to blood group O. The remaining two (2)HBsAg Positive belonged to A1 blood group.

In which both 3rd and 4th generation ELISA test 2 seroreactive samples.

87 Table:27 Rh distribution of seropositive of HCV & HBsAg BY 3rd &

4thGeneration ELISA

3rd generation 4th generation HCV HBsAg HCV HBsAg Rh typing seroreactive seroreactive seroreactive (Total seroreactive (Total (Total (Total donors) donors) donors) donors) Positive 0(351) 3(351) 0(351) 3(351) Negative 0(22) 0(22) 1(22) 0(22) TOTAL 0(373) 3(373) 1(373) 3(373)

0.05 4.55% 4.55% 0.045 0.04 0.035 0.03 0.025 0.02 0.015 0.85% 0.85% 0.85% 0.85% 0.01 0.005 0 0 0 0 0 0 0 seroreactive - seroreactive Total seroreactive - seroreactive Total HCV HBsAg Seroreactive HCV HBsAg Seroreactive 3rd generation 4th generation

Positive Negative

Figure:32 Rh Distribution of seropositive of HCV & HBsAg by 3rd & 4thGeneration ELISA

In the present study Rh positive donors showed 3(351) seropositivity

(0.85%) for HBsAg in both 3rd and 4th generation HBsAg ELISA whereas Rh negative donor showed 1(22) seropositivity (4.55%) only 4th generation HCV

ELISA.

88 Table: 28 Distribution of seropositive of HCV & HBsAg BY 3rd &

4thGeneration ELISA among first time & repeat donors

3rd generation 4th generation HCV HBsAg HCV HBsAg Number of Donation seroreactive seroreactive seroreactive seroreactive (Total (Total (Total donors) (Total donors) donors) donors) First time 0(233) 2(233) 1(233) 2(233) Repeat 0(140) 1(140) 0(140) 1(140) TOTAL 0(373) 3(373) 1(373) 3(373)

0.71% n

o Total Seroreactive 1.29% i t a r

e 0.71% n seroreactive HBsAg 0.86% e g

t 0 4 seroreactive -HCV 0.43%

n Total Seroreactive 0.71% o

i 0.86% t a r

e 0.71% n seroreactive HBsAg 0.86% e g

r 0 3 seroreactive -HCV 0

0 0.002 0.004 0.006 0.008 0.01 0.012 0.014

Repeat First time

Figure:33 Distribution of seropositive of HCV & HBsAg by 3rd &

4thGeneration ELISA AMONG first time & repeat donors

In this study 2 (233) HBsAg seroreactive donations (0.86%) belonged to first time donors and the remaining 1 (140) seroreactive donation (0.71%) belonged to a repeat voluntary blood donation which showed seroreactivity in 3rd & 4th generation HBsAg ELISA, 1(233) HCV seroreactive (0.86%) belonged to first time donation by 4th generation ELISA and did not pick up in 3rdgeneration HCV

ELISA.

89 Table: 29 Seroprevalence

3rd generation 4th generation Reactive Nonreactive Reactive Nonreactive HIV 0 373 0 373 HBsAg 3 370 3 370 HCV 0 373 1 372

SEROPREVALENCE

1.07%

0.012 0.80% 0.01 0.80% 0.80% 0.008 0.006 0.27% 0.004 0 4th generation 0.002 0 0 3rd generation 0 HIV HBsAg HCV TOTAL

3rd generation 4th generation

Figure:34Seroprevalence

In our study, 3rd generation ELISA had 3 donor positive for HBsAg which was 0.80% and no one found positive for HCV. Whereas, 3 donors were positive for HBsAg which was 0.80% and there was 1 donor found to be positive for

HCV which was 0.27% in 4th generation ELISA.

90 Table: 30Real time PCR

3rd generation ELISA 4th generation ELISA RT PCR HCV HBsAg HCV HBsAg HCV HBV POSITIVE 0 3 1 3 1 3 NEGATIVE 1 0 0 0 0 0

3 3 3 3 2.5 2 1.5 1 1 1 1 0.5 0 0 0 0 0 0 0 HCV HBsAg HCV HBsAg HCV HBV 3rd generation ELISA 4th generation ELISA RT PCR POSITIVE NEGATIVE

Figure:35Real time PCR results of seropositive donor

All 3 samples which were HBsAg seroreactive by both 3rd and 4th generation ELISA showed the presence of HBV DNA by RT PCR.

Only one sample was seroreactive by 4th generation ELISA, that too was not detected by 3rd generation ELISA. RT PCR done on this sample revealed the presence of HCV RNA.

Highest Highest SR- No of Seroprevalence 3rd Seroprevalence 4th SR-Age Age intervel Parameters Gender Place of study donors generation ELISA generation ELISA intervel - - 4th Gen 3rd Gen ELISA M F HIV HBsAg HCV HIV HBsAg HCV Present study 373 320(85.8%) 53(14.2% Chennai 0.00% 0.80% 0.00% 0.00% 0.80% 0.27% 21-30 yrs 21-30 yrs Sheetal malhotra et al.,137 10200 Chandigarh-2013 0.13% 0.36%

130

9 kola sujatha et al ., 100 92% 8% India-2017 0.00% 1.00% 22 yrs 2 Arora et al.,136 1839 Haryana-2010 0.00% 1.70% 1.00% 18-31 yrs

Sachin Shivaji kapse.,135 4708 86.6% 13.4% Mumbai-2019 1.10% 0.20% 0.08%

Sangita patel et al.,127 14239 95.40% 4.60% Gujarat-2013 0.21% 0.18% 0.72%

Bridon M' baya.,126 125893 Malawi-2018 1.00% 1.90% 3.60% >25 yrs >25 yrs

makroo et al.,5 New Delhi-2013 0.24% 1.18% 0.21%

Akanksha et al.,138 163540 Delhi-2015 0.32% 1.61% . 0.73%.

Sheetal Bridon M' Solomon Bisetegen Parameters Present study malhotra baya et al.,126 et al.,140 et al.,137 Infection HIV HBsAg HCV HIV HIV HBsAg HCV HIV HBsAg HCV 3rd Gen 0.00% 0.42% 0.00% 0.03% 1.10% Student- seroreactive 4th gen 0.00% 0.42% 0.00% 0.18% 1.40% 3.50%

3rd Gen 0.00% 0.75% 0.75% 0.24% 2.10% Non Student- 10.09 seroreactive 4th gen 0.00% 0.75% 1.50% 0.55% 12.10% %

9 A 2.35% 10.40% 3 Blood Group- B 13.00%

seroreactive AB

O 1.30%

POS 0.85% Rh type- seroreactive NEG 4.55%

Type of Donor- 3rd first time 0.86% 0.16% 1.20%

Gen- seroreactive repeat 0.71% 0.12% 0.90%

Type of Donor- 4th first time 0.86% 2.80% 5.10%

Gen- seroreactive repeat 0.71% 0.43% 1.20% 2.30%

DISCUSSION

In the present cross sectional study, 373 voluntary blood donors were screened to compare the sensitivity of 3rd and 4th generation ELISA kits and also to know the prevalence of HIV, HCV and HBsAg.

Voluntary blood donors with asymptomatic infection of HIV, HCV and HBsAg during window period contribute to the risk of Transfusion

Transmitted infection.

Our blood bank completely depends on voluntary blood donation. All the donors included in the study were voluntary blood donors.

In the present study,320 (85.8%) were males and 53 (14.2%) were females. Similarly, in the study done by Sachin Shivaji kapse et al, 135 86.6% were male donors and 13.4% were female donors. However in the study done by Sangita

Patel et al.127 the overall sex distribution was 95.4% males and 4.6% females.

The main reason for lower female donors were due to high prevalence of anaemia among Indian women and less female participation due to lack of awareness, motivation and education regarding voluntary blood donation. (Ref)

In the present study, the seroreactive of HBsAg and HCV were 0.8%,

0.27% respectively. None of the voluntary blood donors were found to be HIV seroreactive.

Similar study done by Arora et al., 136 among replacement donors in state of Haryana the seroreactive of HBsAg, HCV and HIV were 1.4% , 0.9% and

0.3 % respectively. However, among voluntary blood donors in the same study,

94 they found lower seroreactive for HBsAg and HCV viz., 0.3 and 0.12 % respectively, none were HIV seroreactive.

In the present study HIV seroreactive was estimated to be 0 per 373 donations (0%) using 3rd and 4thgeneration ELISA. In a study by Sheetal Malhotra et al., 137 HIV seroreactive was estimated to be 1.37 per 1000 donations (0.13%) using

3rd generation ELISA and 3.62 per 1000 donations (0.36%) by using the

4th generation ELISA. Their study was also done on only voluntary blood donors similar to our study, however slightly higher seroreactive by 4th generation ELISA was found to be due to higher false positive reactions (33 % of samples found seroreactive by 4th generation ELISA was found to be nonreactive by western blot study).

Kola sujatha et al., 130 in their study among voluntary blood donors from rural population in the state of Andhra Pradesh, found 0% HIV seroreactive by 3rd generation ELISA and 1% by 4th generation ELISA.

In the present study, HBsAg seroreactive was estimated to be same

(0.8%) by both 3rd and 4th Generation ELISA. In a study among blood donors in

Mumbai (both replacement and Voluntary), Sachin Shivaji kapse et al., 135 found

1.1% HBsAg seroreactive by 3rd generation ELISA.

Patel et al., in their study among voluntary blood donors in Gujarat, by

4th generation HBsAg ELISA found 0.63% HBV seroreactive.

In the present study, HCV seroreactive was estimated to be 0 per 373 donations using 3rd generation ELISA and 0.27 % (1 per 373 donors) HCV by 4th generation ELISA.

95 Makroo et al.,5 studyamong blood donors (96.9% replacement and 3.1% voluntary) in New Delhi revealed 0.43% seroreactive by 3rd generation ELISA. Patel et al., study in Gujarat found 0.21% HCV seroreactive by 3rd generation ELISA.

Akanksha et al.,138 in their study among blood donors in New Delhi (25.4% replacement and 74.6% voluntary) found 0.73% HCV seroreactive by 4th generation

ELISA.

The wide variations in the seroreactive of HIV, HCV and HBV among the voluntary blood donors in different studies conducted in India is most probably due to the use of different methods of testing and use of different generation of

ELISA test kits having different sensitivities and specificities.

In the present study, the most common age group of voluntary blood donors were less than 18-20 years (49.1%) and 32.4% in 21-30 years, 9.4% in 31-

40 years, 6.7 % in 41-50 years, 2.4% in >50 Years. However the age distribution of seropositivity among the voluntary blood donors is seen in 21-30 years of age which is 3.3% and these is similar to the following studies,

In the study by Sachin Shivaji kapse et al..,135 Majority of TTI positivity is among 18 – 27 years about 0.59%.(3rd generation). In the study by Ahmed et al.,139 had 69.8% positive donors belonged to the age group of 18-30 years, and

28.3% belong to 31-45 years age group.

In the study conducted by Solomon Bisetegen et al.,140 65 (23.6%) of donors less than or equal to 30 years and 50 (43.9%) of donors greater than 30 years have evidence of at least one blood borne pathogen.(4th generation)

The present study had all seroreactive donation belonged to male gender which implies no female donors had seroreactivity. In which 3 donations

96 belonged to HBV and 1 had HCV positivity. The 3rd generation ELISA showed

3(0.94) out of 4 positive donations and did not pick up 1(0.31%) out of 4HCV positive donation. Whereas 4th generation ELISA picked all 4 (1.25%) out of 4 as positive reactions (3 HBsAg and 1HCV)

In the present study among the total of 373 voluntary blood donations

233 were first time donation and the remaining 140 were repeat donations. In which all3 HBsAg seroreactive donations, 2 (233) HBsAg seroreactive donations (0.86%) belonged to first time donors and the remaining 1 (140) 0.71% seroreactive donation belonged to a repeat voluntary blood donation which showed seroreactivity in 3rd generation ELISA.

However, 3rd generation ELISA did not pick up 1 HCV seroreactive donation. Further, in 4th generation ELISA picked up all 4 /4 seroreactive donations in which 3 HBsAg seroreactive donations, 2 (233) HBsAg seroreactive donations

(0.86%) belonged to first time donation and the remaining 1 (140) 0.71% seroreactive donation belonged to a repeat voluntary blood donation and further, it also picked up 1 seroreactive HCV belonged to first time donation.

Similarly in the study conducted by Sachin Shivaji kapse et al,135 1.01% seroreactivity among 1st time donors and 0.38%seroreactivity in repeat donors the study was done by using 3rd generation ELISA kits.

In the study done by Sheetal Malhotra et al.,137 had 1.61 /1000 1st time donors and, 1.23/1000 repeat donors found seroreactive in 3rd generation ELISA however in 4th generation ELISA 2.94/1000 showed seroreactive results and

0.69/1000 found to be in grey zone in both 1st time and repeat donors.

97 RT-PCR:

In this study, to confirm seropositive samples RT-PCR was done.

All 3 samples which were HBsAg seroreactive by both 3rd and 4th generation ELISA showed the presence of HBV DNA by RT PCR, thereby ruled out any false positive reactions.

Only one sample was seroreactive by 4th generation ELISA, that too was not detected by 3rd generation ELISA. RT PCR done on this sample revealed the presence of HCV RNA (2.8x105IU/ML, Genotype 1b), thereby ruled out false positive reaction by 4th generation ELISA.

The sample which was nonreactive by 3rd generation ELISA found to be reactive by 4th generation ELISA for HCV is due to the ability of 4th generation

ELISA to detect HCV core antigen much earlier than HCV antibody by 3rd generation ELISA. The Syria Laperche et al.,134 study on simultaneous detection

HCV core antigen and anti-HCV antibodies on voluntary blood donors revealed 33 to 46% reduction in the HCV related TTI.

All the four seroreactive donors were given post-test counselling and were advised to refrain from high risk behaviour and also to self-exclude from future donations. All the four seroreactive donors was referred to medical gastroenterology department at a higher centre for counselling, Management and further follow up.

SUMMARY

SUMMARY x This study was conducted to compare detection of HBV, HCV

and HIV among voluntary blood donors in Chennai by 3rd and 4th

generation ELISA. x Among total 373 voluntary blood donor samples, 3 were found to be

seroreactive for HBsAg by 3rd and 4th generation ELISA. Only one

sample was found to be seroreactive for HCV by 4th generation

ELISA, this was found to nonreactive by 3rd generation ELISA. None

of the samples were reactive for HIV by both 3rd and 4th generation

ELISA. x 233 donors were first time donors and the remaining 140 were repeat

donors. x 2 of the first time donor samples and one of the repeat donor sample

were found to be reactive for HBsAg by both 4th and 3rd generation

ELISA. x The sample which was reactive for HCV by 4th generation ELISA was

from a first time blood donor. x The 3 samples which were found to be reactive for HBsAg by 3rd and

4th generation ELISA and only one sample which was reactive for

HCV by 4th generation ELISA were confirmed by RT PCR.

CONCLUSION

In the present study, there is no difference in detection of HBsAg and

HIV between 3rd and 4th generation ELISA. In detection of HCV by 4th generation

ELISA is found to be more sensitive than 3rd generation ELISA.

However, it is imperative to do a study on larger number of samples to arrive at a definitive conclusion.

100

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96. López-Labrador FX, Ampurdanés S, Forns X, Castells A, Sáiz JC, Costa J, et al. Hepatitis C virus (HCV) genotypes in Spanish patients with HCV infection: relationship between genotype 1b, cirrhosis and hepatocellular carcinoma. J Hepatol. 1997;27:959–65.

xi 97. Pouillot R, Lachenal G, Pybus OG, Rousset D, Njouom R. Variable epidemic histories of hepatitis C virus genotype 2 infection in West Africa and Cameroon. Infect Genet Evol. 2008;8:676–81.

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xiii 116. Yorde DE, Sasse EA, Wang TY, Hussa RO, Garancis JC. Competitive enzyme-liked immunoassay with use of soluble enzyme/antibody immune complexesfor labeling. I. Measurement of human choriogonadotropin. Clin Chem1976;22:1372–7.

117. Aydin, S. (2015). A short history, principles, and types of ELISA, and our laboratory experience with peptide/protein analyses using ELISA. Peptides, 72, 4 15. doi:10.1016/j.peptides.2015.04.012.

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120. Lequin RM (2005) Enzyme immunoassay (EIA)/enzyme-linked immunosorbent assay (ELISA). Clin Chem 51: 2415-2418.

121. Bäcker U, Gathof A, Howe J, Strobel E, Deinhardt F, et al. (1988) A new second-generation anti-HIV-1 enzyme immunoassay using recombinant envelope and core proteins. AIDS 2: 477-480.

122. Maskill WJ, Crofts N, Waldman E, Healey DS, Howard TS, et al. (1988) An evaluation of competitive and second generation ELISA screening tests for antibody to HIV. J Virol Methods 22: 61-73.

123. Zaaijer HL, Exel-Oehlers PV, Kraaijeveld T, Altena E, Lelie PN (1992) Early detection of antibodies to HIV-1 by third-generation assays. Lancet 340: 770-772.

124. Vallefuoco L, Mazzarella C, Portella G (2016) Fourth generation assays for HIV testing. Exp Rev Mol Diagn 16: 723-732.

xiv 125. Olowe OA, Mabayoje VO, Akanbi O, et al. HIV P24 antigen among HIV antibody sero-negative blood donors in Osogbo Osun State, South Western Nigeria. Pathog Glob Health. 2016; 10(4-5):205–208. doi:10.1080/20477724.2016.1205311.

126. Seroprevalence and trends in transfusion transmissible infections among voluntary non-remunerated blood donors at the Malawi Blood Transfusion Service-a time trend study M’baya et al; Malawi Medical Journal 31 (2): 118-125 June 2019

127. Sangita Patel et al.,Seroprevalence of HIV, HBV, HCV and syphilis in blood donors at a tertiary hospital (blood bank) in Vadodara.DOI: 10.5455/ijmsph.2013.160520132.

128. Muthukumar, A., Alatoom, A., Burns, S., Ashmore, J., Kim, A., Emerson, B., … Ansari, M. Q. (2015). Comparison of 4th-Generation HIV Antigen/Antibody Combination Assay With 3rd-Generation HIV Antibody Assays for the Occurrence of False-Positive and False-Negative Results. Laboratory Medicine, 46(2), 84–89. doi:10.1309/lmm3x37nswucmvrs

129. Weber B, Fall EH, Berger A, Doerr HW. Reduction of diagnostic window by new fourth-generation human immunodeficiency virus screening assays. J Clin Microbiol. 1998;36(8):2235–2239.

130. Comparative Study of 3rd Generation V/S 4th Generation ELISA in Blood Donors for Early Diagnosis of HIV in Rural Population Kola Sujatha* and Kasi Seshu Vaisakhi RVMIMSRC Medical College, Vaisakhi, Kasi Seshu, VRK, Medical College, India. https://doi.org/10.20546/ijcmas.2017.606.137

131. Comparison of electrochemiluminescence and ELISA methods in the detection ofblood borne pathogens in Gabon Cyrille Bisseye1*, Jophrette, Anicet Mouity Mireille Ntsame Ndong2 Matoumba1, Calixte Bengone2,Guy Mouelet Migolet2, Bolni Marius Nagalo1,31Laboratory of Molecular and Cellular Biology, University of Sciences and Techniques of Masuku (USTM), P.O. Box 943,Franceville, Gabon2National Blood Transfusion Center, P.O. Box 13895, Libreville, Gabon. https://doi.org/ 10.1016/j.apjtb. 2017.08.008

xv 132. Rouet, F., Deleplancque, L., Mboumba, B. B., Sica, J., Mouinga-Ondémé, A., Liégeois, F., Gaudy-Graffin, C. (2015). Usefulness of a Fourth Generation ELISA Assay for the Reliable Identification of HCV Infection in HIV-Positive Adults from Gabon (Central Africa). PLOS ONE, 10(1), e0116975. doi:10.1371/journal.pone.0116975

133. Bjoern Bjoerkvoll et al., screening test accuracy among potential blood donors of HBSAg, Anti- HBc and Anti-HCV to detect Hepatistis B and C virus infection in rural Cambodia and Vietnam.

134. Laperche, S., Le Marrec, N., Girault, A., Bouchardeau, F., Servant-Delmas, A., Maniez-Montreuil, M., … Simon, N. (2005). Simultaneous Detection of Hepatitis C Virus (HCV) Core Antigen and Anti-HCV Antibodies Improves the Early Detection of HCV Infection. Journal of Clinical Microbiology, 43(8), 3877–3883. doi:10.1128/jcm.43.8.3877-3883.2005

135. Sachin Shivaji Kapse, Shanu Srivastava, Aradhana Mishra Seroepidemiology of transfusion-transmitted infections among blood donors at a tertiary care center in Navi Mumbai, Maharashtra

136. Arora D, Arora B, Khetarpal A. Seroprevalence of HIV, HBV, HCV and syphilis in blood donors in Southern Haryana. Indian J Pathol Microbiol 2010;53:308-9

137. Seroprevalence of human immunodeficiency virus in north Indian blood donors using third and fourth generation Enzyme linked immunosorbent assay Sheetal Malhotra, Neelam Marwaha, Karan Saluja

138. Seroprevalence & changing trends of transfusion transmitted infections amongst blood donors in a Regional Blood Transfusion Centre in north India Akanksha Rawat, Preeti Diwaker, Priyanka Gogoi, and Bharat Singh doi: 10.4103/ijmr.IJMR_468_15: 10.4103/ijmr.IJMR_468_15

xvi 139. Seroprevalence of Human Immunodeficiency Virus, Hepatitis B Virus, Hepatitis C Virus, and Syphilis in Blood Donors at District Level Blood Bank in a Teaching Hospital, Mandya, KarnatakaKhalid Ahmed1, K L Shoba2, B Sumangala3, Mamatha P Samaga4, B S Akshantha,1 N S Sahana Shetty1

140. Bisetegen, F. S., Bekele, F. B., Ageru, T. A., & Wada, F. W. (2016). Transfusion-Transmissible Infections among Voluntary Blood Donors at Wolaita Sodo University Teaching Referral Hospital, South Ethiopia. Canadian Journal of Infectious Diseases and Medical Microbiology, 2016, 1–6. doi:10.1155/2016/8254343

141. Eko Mba, J. M., Bisseye, C., Mombo, L. E., Ntsame Ndong, J. M., Mbina Ekayeng, S. C., Bengone, C., … Nagalo, B. M. (2018). Assessment of rapid diagnostic tests and fourth-generation Enzyme-Linked Immunosorbent Assays in the screening of Human Immunodeficiency and Hepatitis B virus infections among first-time blood donors in Libreville (Gabon). Journal of Clinical Laboratory Analysis, e22824. doi:10.1002/jcla.22824

142. Majdabadi HA, Kahouei M, Taslimi S, Langari M. Awareness of and attitude towards blood donation in students at the Semnan University of Medical Sciences. Electron Physician. 2018;10(5):6821–6828. Published 2018 May 5. doi:10.19082/6821.

xvii

ANNEXURES

xviii

xix

CERTIFICATE II

This is to certify that this dissertation work titled “COMPARATIVE

STUDY OF HIV, HCV AND HBV SCREENING BYTHIRD AND FOURTH

GENERATION ELISA IN DONATED BLOOD” of the candidate

Dr.J.VASANTH KUMAR with registration number 201731004 for the award of

MD in the branch of IMMUNOHAEMATOLOGY AND BLOOD

TRANSFUSION. I personally verified urkund.com website for the purpose of plagiarism check. I found that the uploaded thesis file contains from introduction to conclusion pages and result shows 4 percentage of plagiarism in the dissertation.

Guide & Supervisor sign with SEAL

xx DONOR INFORMATION SHEET

COMPARATIVE STUDY OF HIV, HCV AND HBV SCREENING BY THIRD AND FOURTH GENERATION ELISA IN DONATED BLOOD

Transfusion transmitted infection such as HIV, HCV and HBV more common in developing nation. It can be transmitted through transfusion of blood products. It can cause morbidity and mortality in all age groups which could be prevented by screening the blood product.

AIM:

To find out the sensitivity of fourth generation ELISA compare with third generation of ELISA.

PROCEDURE

Being a voluntary blood donor, you are recruited for this study. 10 ml of blood will be taken separately. Your blood sample used for detecting HIV, HCV and HBV by third and fourth generation ELISA method.

BENEFITS AND RISKS

All the investigations done at free of cost.

There is no risk for these investigations.

CONFIDENTIALITY

Your privacy will be protected in so for as permitted by law. Only your researcher and Ethical committee members will have access to the data collected during the study.

PARTICIPATION

Your participation in this study is voluntary and you are free to decide now or later whether to continue or discontinue from the study.

Name of the Donor: Signature: Date:

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xxii DONOR CONSENT FORM

I confirm that I read and understood the information about the above research study dated ______and I received chance to ask the questions.

My participation in this study is voluntary and I know that I am free to withdraw from the study at any time, without giving any reason and without affecting of my legal rights.

I agree to this access. I know that my identification will not be revealed in any details that is released to third persons or published.

I agree not to restrict or interfere with any data or results that are obtained from this study. I agree to participate in this research study for the above listed purpose.

Name of the Donor: Signature: Date:

Signature of the person who obtains consent: Date:

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xxxiv MASTER CHART

3rd Generation ELISA 4th Generation ELISA anti anti- Type S. Donor Age Weight Socioeconomi No. Of Blood Rh Repeat anti HIV HIV I HCV HBsAg Gender Area Zone Occupation of TTI Month anti- no id interval in kg c class donation group TYPE reactive I HBsAg & II and & donor HCV & II and core Variants p24 Ag Ag 1 1034 2 1 81 Pudupet 5 2 2 1 1 2 1 2 8 2 2 2 2 2 2

2 1035 3 1 73 Avanipuram 16 1 2 1 2 1 1 2 8 2 2 2 2 2 2

3 1036 3 1 69 Anna nagar 8 1 2 1 2 3 1 2 8 2 2 2 2 2 2

4 1037 4 1 82 Avanipuram 16 1 2 1 2 3 1 2 8 2 2 2 2 2 2

5 1038 3 1 93 Chinmayanagar 11 1 2 1 2 1 1 2 8 2 2 2 2 2 2

6 1039 1 1 92 Villivakam 8 2 2 1 1 3 1 2 8 2 2 2 2 2 2

7 1040 1 1 72 Vadapalani 10 2 2 1 1 2 1 2 8 2 2 2 2 2 2

8 1041 2 1 62 Parisalai 16 1 3 1 1 1 1 2 8 2 2 2 2 2 2

9 1042 3 1 63 West mambalam 10 1 2 1 1 3 1 2 8 2 2 2 2 2 2

10 1043 2 1 75 Medavakkam 14 1 2 1 1 2 1 2 8 2 2 2 2 2 2

11 1044 5 1 76 Kilpauk 8 1 2 1 2 1 1 2 8 2 2 2 2 2 2

12 1045 2 1 76 Kilpauk 8 2 2 1 2 2 1 2 8 2 2 2 2 2 2

13 1046 3 1 88 Choolaimedu 8 1 2 1 2 2 1 2 8 2 2 2 2 2 2

14 1047 1 1 61 Arumbakam 7 2 2 1 1 4 1 2 8 2 2 2 2 2 2

15 1048 3 1 89 Arumbakam 7 1 3 1 1 2 1 2 8 2 2 2 2 2 2

16 1049 4 1 68 Arumbakam 7 2 2 1 2 1 1 2 8 2 2 2 2 2 2

17 1050 4 1 61 Kelambakkam 16 1 2 1 2 3 1 2 8 2 2 2 2 2 2

18 1051 1 1 65 Thiruvottiyur 1 2 2 1 1 1 1 2 8 2 2 2 2 2 2

19 1052 1 1 77 Korattur 6 2 2 1 1 4 1 2 8 2 2 2 2 2 2

20 1053 1 1 105 Jawahar nagar 11 2 2 1 1 2 1 2 8 2 2 2 2 2 2

21 1054 1 1 65 Sholinganallur 15 2 2 1 1 4 1 2 8 2 2 2 2 2 2

22 1055 1 1 105 Pulinthope 1 2 2 1 1 1 1 2 8 2 2 2 2 2 2

23 1056 1 1 85 Kondithope 16 2 2 1 1 1 2 2 8 2 2 2 2 2 2

24 1057 1 1 95 Seven hills 7 2 2 1 1 3 1 2 8 2 2 2 2 2 2

25 1058 1 1 82 Ennore 1 2 2 1 2 4 1 2 8 2 2 2 2 2 2

26 1059 1 1 86 Teynampet 9 2 2 1 1 1 1 2 8 2 2 2 2 2 2

27 1060 1 1 70 Nanagallur 16 2 2 1 1 1 1 2 8 2 2 2 2 2 2

28 1061 1 1 64 Ennore 1 2 2 1 1 3 1 2 8 2 2 2 2 2 2

29 1062 1 1 60 Kavaraipettai 16 2 2 1 1 1 1 2 8 2 2 2 2 2 2

30 1063 1 1 62 Washermanpet 4 2 2 1 1 1 1 2 8 2 2 2 2 2 2

31 1064 1 1 75 Mellu street 1 2 2 1 2 4 1 2 8 2 2 2 2 2 2

32 1087 1 2 67 Otteri 4 2 2 1 1 3 1 2 8 2 2 2 2 2 2

33 1105 2 1 68 Alandur 12 1 2 1 2 3 1 2 9 2 2 2 2 2 2

34 1107 2 1 75 Thiruvallur 16 1 2 1 2 1 1 2 9 2 2 2 2 2 2

35 1111 1 1 75 Thiruvallur 16 2 2 1 1 2 1 2 9 2 2 2 2 2 2

36 1114 1 1 90 Kattambatthur 16 2 2 1 1 2 1 2 9 2 2 2 2 2 2

37 1115 1 1 68 Purasaivakkam 8 2 2 1 1 3 1 2 9 2 2 2 2 2 2

38 1116 1 1 60 Redhills 7 2 3 1 1 2 2 2 9 2 2 2 2 2 2

39 1117 1 1 80 Thiruvallur 16 2 3 1 1 3 1 2 9 2 2 2 2 2 2

40 1118 1 1 50 Kodungaiyur 4 2 3 1 1 3 2 2 9 2 2 2 2 2 2

41 1128 1 1 75 Thiruvallur 16 2 3 1 1 3 1 2 9 2 2 2 2 2 2

42 1129 1 1 85 Vysarpadi 4 2 2 1 1 3 1 2 9 2 2 2 2 2 2

43 1130 1 1 57 Thiruvallur 16 2 2 1 1 2 1 2 9 2 2 2 2 2 2

44 1131 1 1 53 Sharmanagar 4 2 2 1 1 3 1 2 9 2 2 2 2 2 2

45 1132 1 1 82 Mamballam 9 2 2 1 1 2 1 2 9 2 2 2 2 2 2

46 1142 1 1 57 Mambalam 9 2 2 1 1 3 1 2 9 2 2 2 2 2 2

47 1143 2 1 63 Gandhi nagar 13 2 2 1 1 3 1 2 9 2 2 2 2 2 2

48 1144 2 1 62 Selavayal 5 2 2 1 2 3 1 2 9 2 2 2 2 2 2

49 1145 1 1 65 Vyasarpadi 4 2 2 1 1 3 1 2 9 2 2 2 2 2 2

50 1146 1 1 75 Manali 2 2 2 1 1 2 1 2 9 2 2 2 2 2 2

51 1156 1 1 85 Vysarpadi 4 2 2 1 1 2 2 2 9 2 2 2 2 2 2

52 1157 1 1 55 Vysarpadi 4 2 2 1 1 3 1 2 9 2 2 2 2 2 2

53 1158 1 1 60 Kodungaiyur 4 2 2 1 1 3 2 2 9 2 2 2 2 2 2

54 1159 1 1 55 Vysarpadi 4 2 2 1 2 3 1 2 9 2 2 2 2 2 2

55 1160 1 1 53 Thiruvattiyur 1 2 2 1 1 1 1 2 9 2 2 2 2 2 2

56 1170 2 2 80 Chrompet 14 1 2 1 1 2 1 2 9 2 2 2 2 2 2

57 1171 3 1 71 Porur 11 1 2 1 2 3 1 2 9 2 2 2 2 2 2

58 1172 4 1 75 Tambaram 10 1 3 1 2 1 1 2 9 2 2 2 2 2 2

59 1173 2 1 67 Sivagangai 16 1 2 1 1 2 1 2 9 2 2 2 2 2 2

60 1174 2 1 86 West thambaram 10 1 2 1 1 3 1 2 9 2 2 2 2 2 2

61 1194 2 1 60 Tambaran 10 1 2 1 1 3 2 1 1 9 2 2 2 2 1 2 62 1239 1 2 50 Nellore 16 2 2 1 1 3 1 2 10 2 2 2 2 2 2

63 1240 1 1 60 Manaparai 16 2 2 1 1 1 1 2 10 2 2 2 2 2 2

64 1241 1 1 76 Chinmayanagar 11 2 2 1 1 3 1 2 10 2 2 2 2 2 2

65 1242 1 2 91 Kilpauk 8 2 2 1 1 3 1 2 10 2 2 2 2 2 2

66 1243 1 1 78 Kanchipuram 16 2 2 1 1 2 1 2 10 2 2 2 2 2 2

67 1244 2 1 64 Guduvancherry 14 2 2 1 2 3 1 2 10 2 2 2 2 2 2

68 1245 1 1 70 Kanchipuram 16 2 2 1 1 2 1 2 10 2 2 2 2 2 2

69 1246 1 1 54 Urapakkam 16 2 2 1 1 1 1 2 10 2 2 2 2 2 2

70 1247 1 1 60 Chrompet 14 2 3 1 1 3 2 2 10 2 2 2 2 2 2

71 1248 1 1 55 Tambaram 14 2 3 1 1 3 1 2 10 2 2 2 2 2 2

72 1249 1 1 52 Tondiarpet 4 2 3 1 1 3 1 2 10 2 2 2 2 2 2

73 1250 1 1 71 Chrompet 14 2 3 1 1 2 1 2 10 2 2 2 2 2 2

74 1259 1 1 61 Mgr nagar 11 2 2 1 1 4 1 2 10 2 2 2 2 2 2

75 1260 1 1 59 Mamballam 10 2 2 1 1 2 1 2 10 2 2 2 2 2 2

76 1261 1 1 64 Chengalpattu 16 2 2 1 1 2 1 2 10 2 2 2 2 2 2

77 1262 1 1 59 Perungulattur 14 2 2 1 1 4 1 2 10 2 2 2 2 2 2

78 1263 2 1 108 Ashok nagar 12 2 2 1 1 3 1 2 10 2 2 2 2 2 2

79 1264 1 1 56 Ouducherry 16 2 2 1 1 2 1 2 10 2 2 2 2 2 2

80 1265 1 2 55 Pozhichur 14 3 2 1 1 3 1 2 10 2 2 2 2 2 2

81 1266 1 2 57 Gophamedu 1 2 2 1 1 1 1 2 10 2 2 2 2 2 2

82 1267 1 2 57 Serappacherry 1 2 2 1 1 2 1 2 10 2 2 2 2 2 2

83 1268 1 2 66 Iyancherry 1 2 2 1 1 3 1 2 10 2 2 2 2 2 2

84 1331 2 1 67 Thambaram 14 2 2 1 1 2 1 2 10 2 2 2 2 2 2

85 1332 2 2 80 Thalambur 15 2 2 1 1 3 1 2 10 2 2 2 2 2 2

86 1333 2 1 94 Thalambur 15 3 2 1 1 2 1 2 10 2 2 2 2 2 2

87 1334 2 1 70 Puttaparthy 16 2 2 1 2 3 1 2 10 2 2 2 2 2 2

88 1335 1 1 95 Vellore 16 2 2 1 1 3 1 2 10 2 2 2 2 2 2

89 1336 2 2 75 Anjugam nagar 14 2 2 1 1 3 1 2 10 2 2 2 2 2 2

90 1337 1 1 90 Nandigramam 13 2 2 1 1 1 1 2 10 2 2 2 2 2 2

91 1369 1 1 68 Thiruvallur 16 2 2 1 2 3 1 2 11 2 2 2 2 2 2

92 1370 1 1 65 Villupuram 16 2 2 1 1 3 2 2 11 2 2 2 2 2 2

93 1371 2 1 55 Thiruvallur 16 2 3 1 1 3 2 2 11 2 2 2 2 2 2

94 1372 1 2 68 Selaiyur 1 2 3 1 1 1 1 2 11 2 2 2 2 2 2

95 1373 1 2 70 KK nagar 11 2 3 1 1 4 1 2 11 2 2 2 2 2 2

96 1374 1 2 70 KK nagar 11 2 3 1 1 5 1 2 11 2 2 2 2 2 2

97 1375 1 1 58 Kuntrathur 11 2 3 1 1 2 1 2 11 2 2 2 2 2 2

98 1376 1 2 67 Anakauthur 12 2 2 1 1 2 1 2 11 2 2 2 2 2 2

99 1377 1 1 62 Poonamalle 7 3 2 1 1 3 1 2 11 2 2 2 2 2 2

100 1378 2 1 55 Kumbakonam 16 2 2 1 2 1 1 2 11 2 2 2 2 2 2

101 1392 1 2 56 paLlavaram 14 2 2 1 1 1 1 2 11 2 2 2 2 2 2

102 1393 1 1 55 Chetpet 8 2 2 1 1 1 1 2 11 2 2 2 2 2 2

103 1394 1 1 73 Nesapakkam 11 3 2 1 1 1 1 2 11 2 2 2 2 2 2

104 1395 1 2 58 Nesapakkam 11 2 2 1 1 1 1 2 11 2 2 2 2 2 2

105 1396 2 1 95 Alwathirunagar 11 2 2 1 1 1 1 2 11 2 2 2 2 2 2

106 1397 2 1 70 Chrompet 14 3 2 1 1 3 2 2 11 2 2 2 2 2 2

107 1398 2 1 65 Maduravoyal 11 2 2 1 1 2 1 2 11 2 2 2 2 2 2

108 1399 1 2 75 Pattabiram 7 2 2 1 1 3 1 2 11 2 2 2 2 2 2

109 1400 1 2 55 Pernambut 16 2 2 1 1 3 1 2 11 2 2 2 2 2 2

110 1401 1 2 54 Gandhi nagar 13 2 2 1 1 2 1 2 11 2 2 2 2 2 2

111 1402 1 2 64 Chikarayapuram 11 2 2 1 1 3 1 2 11 2 2 2 2 2 2

112 1412 1 2 62 Vellore 16 2 2 1 1 2 1 2 11 2 2 2 2 2 2

113 1413 1 1 73 Kodungaiyur 4 2 2 1 1 3 1 2 11 2 2 2 2 2 2

114 1414 1 1 80 Korukkpet 4 2 2 1 1 3 1 2 11 2 2 2 2 2 2

115 1415 1 1 80 Anakapothur 11 2 2 1 1 3 1 2 11 2 2 2 2 2 2

116 1416 1 1 62 Porur 11 2 2 1 1 3 1 2 11 2 2 2 2 2 2

117 1417 1 1 63 Porur 11 2 2 1 1 2 1 2 11 2 2 2 2 2 2

118 1418 4 1 70 Amajikarai 8 2 2 1 2 2 1 2 11 2 2 2 2 2 2

119 1419 4 2 65 Chennai 1 1 2 1 2 1 1 2 11 2 2 2 2 2 2

120 1420 3 2 85 Poonamalle 7 1 2 1 1 1 1 2 11 2 2 2 2 2 2

121 1421 3 2 69 Noombal 1 1 2 1 1 2 1 2 11 2 2 2 2 2 2

122 1428 2 1 87 Mogapair 7 1 2 1 2 1 1 2 12 2 2 2 2 2 2

123 1429 3 1 100 Mogapair 7 1 2 1 2 1 1 2 12 2 2 2 2 2 2

124 1430 2 1 90 Mogapair 7 1 2 1 2 1 1 2 12 2 2 2 2 2 2

125 1431 2 1 58 Mogapair 7 2 2 1 1 2 1 2 12 2 2 2 2 2 2

126 1432 1 1 69 Mogapair 7 2 2 1 1 2 1 2 12 2 2 2 2 2 2

127 1433 2 1 86 Mogapair 7 2 2 1 2 4 1 2 12 2 2 2 2 2 2

128 1434 3 1 80 Mogapair 7 1 2 1 2 4 1 2 12 2 2 2 2 2 2

129 1435 2 1 77 Mogapair 7 3 2 1 1 1 1 2 12 2 2 2 2 2 2

130 1436 2 2 63 Mogapair 7 2 2 1 2 3 1 2 12 2 2 2 2 2 2

131 1437 1 2 79 Mogapair 7 2 2 1 2 3 1 2 12 2 2 2 2 2 2

132 1441 2 1 59 Thirunelveli 16 2 2 1 2 4 1 2 12 2 2 2 2 2 2

133 1442 2 1 78 Rettipalayam 16 2 2 1 1 1 1 2 12 2 2 2 2 2 2

134 1443 2 1 71 Mogapair 7 2 2 1 1 1 1 2 12 2 2 2 2 2 2

135 1444 5 1 89 Guruusamy road 7 1 2 1 2 2 1 2 12 2 2 2 2 2 2

136 1445 2 2 75 Thoothkudi dt 16 2 2 1 1 3 2 2 12 2 2 2 2 2 2

137 1446 2 1 58 Mogapair 7 2 2 1 1 2 1 2 12 2 2 2 2 2 2

138 1447 3 1 63 Nolambur 7 1 2 1 2 3 1 2 12 2 2 2 2 2 2

139 1448 1 2 75 Nolambur 7 2 2 1 1 3 1 2 12 2 2 2 2 2 2

140 1449 4 1 90 Mogapair 7 1 2 1 2 1 2 2 12 2 2 2 2 2 2

141 1450 3 1 83 Mogapair 7 1 2 1 1 4 1 2 12 2 2 2 2 2 2

142 1451 2 1 75 Mogapair 7 2 2 1 1 3 1 2 12 2 2 2 2 2 2

143 1467 5 1 69 Rajaji salai 9 1 2 1 2 2 1 2 12 2 2 2 2 2 2

144 1468 4 1 Avadi 7 1 2 1 1 2 1 2 12 2 2 2 2 2 2

145 1469 1 1 69 Adyar 13 2 2 1 1 1 1 2 12 2 2 2 2 2 2

146 1470 4 1 60 Kelambakkam 16 1 2 1 2 3 1 2 12 2 2 2 2 2 2

147 1471 2 1 72 Rajaji salai 9 1 2 1 2 1 1 2 12 2 2 2 2 2 2

148 1472 3 1 86 Adyar 13 1 3 1 2 3 1 2 12 2 2 2 2 2 2

149 1473 3 1 70 Vellore 16 1 3 1 2 3 1 2 12 2 2 2 2 2 2

150 1474 2 1 85 Arakonam 16 1 2 1 2 3 1 2 12 2 2 2 2 2 2

151 1475 2 1 65 Pudupakkam 14 1 2 1 2 2 1 2 12 2 2 2 2 2 2

152 1476 2 1 62 Mumbai 16 1 2 1 2 2 1 2 12 2 2 2 2 2 2

153 1477 2 1 70 T nagar 9 1 2 1 2 1 1 2 12 2 2 2 2 2 2

154 1478 3 1 75 Ramapuram 11 1 2 1 2 2 1 2 12 2 2 2 2 2 2

155 1479 2 1 85 Avadi 7 1 2 1 2 2 1 2 12 2 2 2 2 2 2

156 1 2 1 73 Salem 16 1 2 1 2 3 1 2 1 2 2 2 2 2 2

157 2 2 1 76 Nesapakkam 11 1 2 1 2 2 1 2 1 2 2 2 2 2 2

158 3 3 1 92 Perumbakkam 14 1 2 1 2 3 1 2 1 2 2 2 2 2 2

159 16 2 1 74 Thambaram 14 1 2 1 2 3 1 2 1 2 2 2 2 2 2

160 17 2 1 77 Thambaram 14 1 2 1 1 2 1 2 1 2 2 2 2 2 2

161 18 2 1 78 Thambaram 14 1 2 1 1 4 1 2 1 2 2 2 2 2 2

162 19 3 1 105 Thambaram 14 1 2 1 2 2 2 2 1 2 2 2 2 2 2

163 20 2 1 85 Thambaram 14 1 2 1 2 1 1 2 1 2 2 2 2 2 2

164 21 2 1 60 Thambaram 14 1 2 1 2 2 1 2 1 2 2 2 2 2 2

165 22 2 1 86 Thambaram 14 1 2 1 2 3 1 2 1 2 2 2 2 2 2

166 23 2 1 59 Thambaram 14 1 2 1 1 1 1 2 1 2 2 2 2 2 2

167 24 2 1 76 Thambaram 14 1 2 1 1 4 1 2 1 2 2 2 2 2 2

168 25 2 1 50 Thambaram 14 1 2 1 1 2 1 2 1 2 2 2 2 2 2

169 26 2 1 72 Thambaram 14 1 2 1 2 2 1 2 1 2 2 2 2 2 2

170 27 2 1 50 Thambaram 14 1 2 1 1 3 1 2 1 2 2 2 2 2 2

171 28 2 1 60 Thambaram 14 1 2 1 1 1 1 2 1 2 2 2 2 2 2

172 29 2 1 55 Thambaram 14 1 2 1 1 3 1 2 1 2 2 2 2 2 2

173 30 2 1 53 Thambaram 14 1 2 1 1 3 1 2 1 2 2 2 2 2 2

174 54 3 1 78 Sholinganallur 15 1 2 1 2 1 1 2 1 2 2 2 2 2 2

175 55 3 1 58 Sholinganallur 15 1 2 1 2 4 1 2 1 2 2 2 2 2 2

176 56 2 1 95 Sholinganallur 15 1 2 1 1 1 1 2 1 2 2 2 2 2 2

177 57 2 1 77 Sholinganallur 15 1 2 1 2 2 1 2 1 2 2 2 2 2 2

178 58 2 1 55 Sholinganallur 15 1 2 1 1 2 1 2 1 2 2 2 2 2 2

179 59 4 1 76 Sholinganallur 15 1 2 1 2 1 1 2 1 2 2 2 2 2 2

180 60 2 1 62 Ekkatuthangal 13 1 2 1 2 3 1 2 1 2 2 2 2 2 2

181 61 2 1 70 Thirunelveli 16 1 2 1 2 3 1 2 1 2 2 2 2 2 2

182 62 2 1 74 Vellore 16 1 2 1 2 2 1 2 1 2 2 2 2 2 2

183 63 2 1 74 Virudhunagar 16 1 2 1 2 3 1 2 1 2 2 2 2 2 2

184 64 2 1 63 Erode 16 1 2 1 1 3 1 2 1 2 2 2 2 2 2

185 65 3 1 73 Sholinganallur 15 1 2 1 2 3 1 2 1 2 2 2 2 2 2

186 66 2 1 62 Arumbakam 15 1 2 1 2 2 1 2 1 2 2 2 2 2 2

187 67 3 1 63 Sholinganallur 15 1 2 1 2 3 1 2 1 2 2 2 2 2 2

188 68 2 1 65 Sholinganallur 15 1 2 1 2 4 1 2 1 2 2 2 2 2 2

189 69 3 1 65 Sholinganallur 15 1 2 1 1 4 1 2 1 2 2 2 2 2 2

190 70 3 1 80 Nanganallur 12 1 2 1 2 3 1 2 1 2 2 2 2 2 2

191 155 2 1 90 Porur 11 1 2 1 1 3 1 1 1 2 2 2 1 2 2 1 192 185 1 1 85 Thambaram 14 2 2 1 1 3 1 2 2 2 2 2 2 2 2

193 186 1 1 78 Chrompet 14 2 2 1 1 3 1 2 2 2 2 2 2 2 2

194 187 1 1 55 Madipakkam 14 2 2 1 1 3 1 2 2 2 2 2 2 2 2

195 188 1 1 54 Madipakkam 14 2 2 1 1 3 1 2 2 2 2 2 2 2 2

196 189 1 1 99 Perungulattur 14 2 2 1 2 3 1 2 2 2 2 2 2 2 2

197 190 1 1 62 West Thambaram 14 2 2 1 1 2 2 2 2 2 2 2 2 2 2

198 191 1 1 96 Velacheri 14 2 2 1 1 3 1 2 2 2 2 2 2 2 2

199 192 1 1 94 Kelkatalai 14 2 2 1 1 3 1 2 2 2 2 2 2 2 2

200 193 1 1 94 Thambaram 14 2 2 1 1 1 1 2 2 2 2 2 2 2 2

201 194 1 1 94 Mappedu 14 2 2 1 1 1 1 2 2 2 2 2 2 2 2

202 195 1 1 71 West thambaram 14 2 2 1 2 2 1 2 2 2 2 2 2 2 2

203 196 1 1 72 Chitlapakam 14 2 2 1 1 2 1 2 2 2 2 2 2 2 2

204 216 1 1 68 Kelkatalai 14 2 2 1 2 4 1 2 2 2 2 2 2 2 2

205 217 1 1 51 Oorapakkam 14 2 2 1 1 2 1 2 2 2 2 2 2 2 2

206 218 1 1 60 Saidapet 10 2 2 1 1 1 1 2 2 2 2 2 2 2 2

207 219 1 1 75 Saidapet 10 2 2 1 1 3 1 2 2 2 2 2 2 2 2

208 220 1 1 85 West thambaram 14 2 2 1 1 1 1 2 2 2 2 2 2 2 2

209 221 1 1 85 West thambaram 14 2 2 1 1 3 1 2 2 2 2 2 2 2 2

210 222 1 1 75 Chitalapakkam 14 2 2 1 1 2 1 2 2 2 2 2 2 2 2

211 223 1 1 84 Anakapothur 14 2 3 1 1 3 1 2 2 2 2 2 2 2 2

212 224 1 1 86 Agaram 14 2 2 1 1 3 1 2 2 2 2 2 2 2 2

213 225 1 1 68 Thambaram 14 2 2 1 1 2 1 2 2 2 2 2 2 2 2

214 226 1 1 58 Kundrathur 11 2 2 1 2 4 1 2 2 2 2 2 2 2 2

215 227 1 1 57 Chengalpattu 16 2 2 1 1 3 1 2 2 2 2 2 2 2 2

216 228 1 1 73 Medavakkam 14 2 2 1 1 1 1 2 2 2 2 2 2 2 2

217 229 1 1 59 Madipakkam 14 2 2 1 1 4 1 2 2 2 2 2 2 2 2

218 230 1 1 64 Mangadu 11 2 2 1 1 3 1 2 2 2 2 2 2 2 2

219 231 1 1 60 Saidapet 13 2 2 1 1 2 1 2 2 2 2 2 2 2 2

220 290 1 1 71 Kundrathur 11 2 2 1 1 3 1 2 3 2 2 2 2 2 2

221 291 2 1 74 G Y nmc chennai 10 2 2 1 1 3 1 2 3 2 2 2 2 2 2

222 292 1 1 64 Chengalpattu 16 2 2 1 1 3 2 2 3 2 2 2 2 2 2

223 293 2 1 54 Arumbakkam 8 2 2 1 1 1 1 2 3 2 2 2 2 2 2

224 294 1 1 88 Nanganallur 8 2 2 1 1 1 1 2 3 2 2 2 2 2 2

225 295 1 1 93 Arumbakam 8 2 2 1 1 3 1 2 3 2 2 2 2 2 2

226 296 1 2 60 Arumbakam 8 2 2 1 1 2 1 2 3 2 2 2 2 2 2

227 297 2 2 56 Arumbakam 8 2 2 1 1 1 1 2 3 2 2 2 2 2 2

228 298 1 2 62 Arumbakam 8 2 2 1 1 3 1 2 3 2 2 2 2 2 2

229 299 1 1 58 Arumbakam 8 2 2 1 1 1 1 2 3 2 2 2 2 2 2

230 300 2 1 118 Salem 16 2 2 1 1 2 1 2 3 2 2 2 2 2 2

231 301 1 2 89 Arumbakam 8 2 2 1 1 3 1 2 3 2 2 2 2 2 2

232 302 3 2 50 Minjur 1 1 2 1 1 2 1 2 3 2 2 2 2 2 2

233 350 2 1 68 Rathinamangalam 14 2 2 1 2 3 1 2 3 2 2 2 2 2 2

234 351 1 1 79 Rathinamangalam 14 2 2 1 1 3 1 2 3 2 2 2 2 2 2

235 352 1 1 64 Rathinamangalam 14 2 2 1 2 1 1 2 3 2 2 2 2 2 2

236 353 3 1 103 Pallavaram 14 1 2 1 2 3 1 2 3 2 2 2 2 2 2

237 354 2 1 59 Anagabuthur 14 2 2 1 1 4 1 2 3 2 2 2 2 2 2

238 355 2 1 63 Thirukalapur 16 1 2 1 2 3 1 2 3 2 2 2 2 2 2

239 356 5 1 77 Alandur 12 1 2 1 2 1 1 2 3 2 2 2 2 2 2

240 357 2 1 71 Puzuthivakkam 14 1 2 1 1 2 2 2 3 2 2 2 2 2 2

241 358 2 1 82 Alandur 12 2 2 1 2 3 1 2 3 2 2 2 2 2 2

242 359 4 1 105 Alandur 12 1 2 1 2 4 1 2 3 2 2 2 2 2 2

243 360 4 1 90 Alandur 12 1 2 1 2 2 1 2 3 2 2 2 2 2 2

244 361 4 1 76 Alandur 12 1 2 1 2 3 1 2 3 2 2 2 2 2 2

245 362 4 1 74 Adambakkam 12 1 2 1 2 2 1 2 3 2 2 2 2 2 2

246 363 4 1 75 Alandur 12 1 2 1 2 2 1 2 3 2 2 2 2 2 2

247 395 4 2 54 Adambakkam 12 1 2 1 1 1 1 2 3 2 2 2 2 2 2

248 396 2 2 70 Alandur 12 1 2 1 1 2 1 2 3 2 2 2 2 2 2

249 397 2 1 70 Alandur 12 1 2 1 2 3 1 2 3 2 2 2 2 2 2

250 398 3 1 72 Adambakkam 12 1 2 1 2 3 1 2 3 2 2 2 2 2 2

251 399 2 1 94 Alandur 12 1 2 1 2 1 1 2 3 2 2 2 2 2 2

252 400 2 1 86 Manjupuram 16 2 2 1 2 3 2 2 3 2 2 2 2 2 2

253 401 4 1 67 Maduvangarai 12 1 2 1 2 2 1 2 3 2 2 2 2 2 2

Cheran 254 402 2 1 52 16 2 2 1 2 1 1 2 3 2 2 2 2 2 2 mahadevan 255 403 2 1 71 Alandur 12 2 2 1 2 2 1 2 3 2 2 2 2 2 2

256 404 1 1 105 Polichalur 14 2 2 1 1 3 1 2 3 2 2 2 2 2 2

257 405 1 1 63 Anagabuthur 14 2 2 1 1 2 1 2 3 2 2 2 2 2 2

258 406 1 1 63 Adambakkam 12 2 2 1 1 1 1 2 3 2 2 2 2 2 2

259 407 1 1 55 Alandur 12 2 2 1 1 1 1 2 3 2 2 2 2 2 2

260 417 2 1 89 Porur 11 2 2 1 1 1 1 1 1 3 2 2 1 2 2 1 261 420 2 2 58 Kanchipuram 16 2 2 1 1 3 1 2 4 2 2 2 2 2 2

Ponniamman 262 421 2 2 75 11 2 2 1 1 3 1 2 4 2 2 2 2 2 2 medu 263 422 2 2 61 Mogapair 7 2 2 1 1 4 1 2 4 2 2 2 2 2 2

264 423 2 2 60 Kodambakkam 10 2 2 1 1 3 1 2 4 2 2 2 2 2 2

265 424 2 2 51 Thiruporur 16 2 2 1 1 2 1 2 4 2 2 2 2 2 2

266 425 2 2 59 Nammalwarpet 11 2 2 1 1 3 2 2 4 2 2 2 2 2 2

267 426 2 2 58 Royapettah 9 2 2 1 1 2 1 2 4 2 2 2 2 2 2

268 427 2 2 55 Karunilam 6 2 2 1 1 2 1 2 4 2 2 2 2 2 2

269 428 1 2 51 Mylapore 5 2 2 1 1 2 1 2 4 2 2 2 2 2 2

270 429 2 2 59 Arumuganeri 11 2 2 1 1 1 1 2 4 2 2 2 2 2 2

271 430 2 2 72 West mambalam 14 2 2 1 1 3 1 2 4 2 2 2 2 2 2

272 431 1 2 58 Nandhanam 7 2 2 1 1 3 1 2 4 2 2 2 2 2 2

273 432 2 1 65 Vadapalani 10 2 2 1 1 4 1 2 4 2 2 2 2 2 2

274 471 2 1 72 Adyar 13 2 2 1 2 1 1 2 4 2 2 2 2 2 2

275 472 1 1 83 Koturpuram 10 2 2 1 1 3 1 2 4 2 2 2 2 2 2

276 473 2 1 55 Guindy 13 2 2 1 2 3 1 2 4 2 2 2 2 2 2

277 474 1 1 98 Koturpuram 10 2 2 1 2 3 1 2 4 2 2 2 2 2 2

278 475 1 1 65 Adyar 13 2 2 1 1 2 1 2 4 2 2 2 2 2 2

279 476 2 1 80 Nanganallur 11 2 2 1 2 3 1 2 4 2 2 2 2 2 2

280 477 2 2 58 Adyar 13 2 2 1 1 2 1 2 4 2 2 2 2 2 2

281 478 2 1 58 Adyar 13 2 2 1 2 1 1 2 4 2 2 2 2 2 2

282 479 4 1 75 Adyar 13 1 2 1 2 2 2 2 4 2 2 2 2 2 2

283 480 3 1 75 Adyar 13 1 2 1 2 4 1 2 4 2 2 2 2 2 2

284 481 3 1 68 Koturpuram 10 1 2 1 2 1 1 2 4 2 2 2 2 2 2

285 482 4 1 60 Adyar 13 1 2 1 2 2 1 2 4 2 2 2 2 2 2

286 483 4 1 76 Adyar 13 1 2 1 2 2 1 2 4 2 2 2 2 2 2

287 484 3 1 65 Kottur 10 1 2 1 2 2 1 2 4 2 2 2 2 2 2

288 485 2 1 70 Adyar 13 2 2 1 2 3 1 2 4 2 2 2 2 2 2

289 486 3 1 60 Kottur 10 1 2 1 2 1 1 2 4 2 2 2 2 2 2

290 487 5 1 80 Adyar 13 1 2 1 2 2 1 2 4 2 2 2 2 2 2

291 496 2 1 111 Triplicane 16 1 2 1 2 1 1 2 5 2 2 2 2 2 2

292 497 2 1 55 Sankarapuram 16 2 2 1 2 3 1 2 5 2 2 2 2 2 2

293 498 2 1 80 Villupuram 16 2 2 1 1 2 1 2 5 2 2 2 2 2 2

294 499 2 1 76 Saidapet 10 2 2 1 2 2 1 2 5 2 2 2 2 2 2

295 500 2 1 62 Anakaputtur 14 1 1 1 2 1 1 2 5 2 2 2 2 2 2

296 501 4 1 66 KK nagar 11 1 1 1 2 3 2 2 5 2 2 2 2 2 2

297 502 1 1 64 Trichy 16 2 2 1 1 3 1 2 5 2 2 2 2 2 2

298 503 5 1 86 Asthinapuram 14 1 1 1 2 5 1 2 5 2 2 2 2 2 2

299 504 2 1 56 Tirupur 16 2 2 1 1 1 1 2 5 2 2 2 2 2 2

300 505 2 1 85 Thiruvallur 16 1 1 1 2 2 1 2 5 2 2 2 2 2 2

301 588 1 1 98 Katpadi 16 2 2 1 1 3 1 2 6 2 2 2 2 2 2

302 589 1 1 84 Nerkundram 11 2 2 1 2 1 1 2 6 2 2 2 2 2 2

303 590 1 1 63 Ambur 16 2 2 1 1 3 1 2 6 2 2 2 2 2 2

304 591 1 1 84 Alwarthirunagar 11 2 2 1 1 3 1 2 6 2 2 2 2 2 2

305 592 1 1 85 Maduravoyal 11 2 2 1 2 2 1 2 6 2 2 2 2 2 2

306 593 1 1 70 Maduravoyal 11 2 2 1 1 2 1 2 6 2 2 2 2 2 2

307 594 1 1 68 Villupuram 16 2 2 1 1 2 1 2 6 2 2 2 2 2 2

308 595 1 1 76 Mudravoyil 11 2 2 1 1 3 1 2 6 2 2 2 2 2 2

309 596 1 1 54 Gummidipondi 16 2 2 1 2 3 1 2 6 2 2 2 2 2 2

310 597 1 1 51 Nesapakkam 11 2 2 1 1 1 1 2 6 2 2 2 2 2 2

311 598 1 1 63 Chengam 16 2 2 1 1 1 1 2 6 2 2 2 2 2 2

312 599 1 1 85 Vellore 16 2 2 1 1 2 1 2 6 2 2 2 2 2 2

313 600 1 1 54 Kallakurichi 16 2 2 1 1 6 1 2 6 2 2 2 2 2 2

314 601 1 1 55 Thiruverkkadu 16 2 2 1 2 3 1 2 6 2 2 2 2 2 2

315 602 1 1 60 Vallur 16 2 2 1 1 2 1 2 6 2 2 2 2 2 2

316 603 1 1 54 Trichy 16 2 2 1 1 2 1 2 6 2 2 2 2 2 2

317 604 1 1 54 Maduravoyal 11 2 2 1 1 3 1 2 6 2 2 2 2 2 2

318 605 1 1 51 Moolakadai 4 2 2 1 2 3 1 2 6 2 2 2 2 2 2

319 606 1 1 63 Vellore 16 2 2 1 2 4 1 2 6 2 2 2 2 2 2

320 607 1 1 64 Thiruvallur 16 2 2 1 1 3 1 2 6 2 2 2 2 2 2

321 608 1 1 75 Thiruvallur 16 2 2 1 1 3 1 2 6 2 2 2 2 2 2

322 609 1 1 73 Ambur 16 2 2 1 1 3 1 2 6 2 2 2 2 2 2

323 610 1 1 62 Villupuram 16 2 2 1 1 1 1 2 6 2 2 2 2 2 2

324 611 1 1 92 Ambur 16 2 2 1 1 3 1 2 6 2 2 2 2 2 2

325 612 1 1 65 Avadi 7 2 2 1 1 2 1 2 6 2 2 2 2 2 2

326 613 1 1 70 Anakaputtur 16 2 2 1 1 1 1 2 6 2 2 2 2 2 2

327 614 1 1 77 Redhills 16 2 2 1 1 3 1 2 6 2 2 2 2 2 2

328 615 1 1 75 Thiruvallur 16 2 2 1 1 1 1 2 6 2 2 2 2 2 2

329 616 1 1 94 Ambur 16 2 2 1 2 1 1 2 6 2 2 2 2 2 2

330 617 1 1 Mugalivakkam 16 3 2 1 2 1 2 2 6 2 2 2 2 2 2

331 618 3 1 84 Kattupakkam 16 1 1 1 2 2 1 2 6 2 2 2 2 2 2

332 619 4 1 76 Naganallur 12 1 1 1 2 3 1 2 6 2 2 2 2 2 2

333 662 2 1 74 Puzhal 7 1 2 1 2 1 1 1 1 6 2 2 1 2 2 1 334 791 3 1 78 Nerkundram 3 1 1 1 2 3 1 2 7 2 2 2 2 2 2

335 792 5 1 103 Broadway 1 1 1 1 2 3 1 2 7 2 2 2 2 2 2

336 793 2 2 82 Nerkundram 3 1 1 1 1 3 1 2 7 2 2 2 2 2 2

337 794 4 2 55 Tondiarpet 4 1 1 1 1 1 1 2 7 2 2 2 2 2 2

338 795 3 1 77 Anna nagar 8 1 1 1 2 2 1 2 7 2 2 2 2 2 2

339 796 4 1 70 Anna nagar 8 1 1 1 1 2 1 2 7 2 2 2 2 2 2

340 797 5 2 90 Anna nagar 8 1 1 1 2 3 1 2 7 2 2 2 2 2 2

341 798 2 1 65 Pondicherry 16 2 2 1 1 3 1 2 7 2 2 2 2 2 2

342 799 2 2 70 Aynavaram 10 2 2 1 1 2 1 2 7 2 2 2 2 2 2

343 800 5 2 78 Bangalore 16 1 1 1 1 2 1 2 7 2 2 2 2 2 2

344 801 1 1 83 Mayilladurai 16 2 2 1 2 3 1 2 7 2 2 2 2 2 2

345 802 1 1 80 Ponnamalle 11 2 2 1 2 1 1 2 7 2 2 2 2 2 2

346 803 1 1 63 Chindatripet 5 2 2 1 1 3 1 2 7 2 2 2 2 2 2

347 804 1 1 75 Maduravoyal 11 2 2 1 1 3 1 2 7 2 2 2 2 2 2

348 805 2 1 80 Choolaimedu 8 2 2 1 2 3 1 2 7 2 2 2 2 2 2

349 806 2 1 63 Puzhal 3 1 1 1 2 3 2 2 7 2 2 2 2 2 2

350 807 2 1 102 Saligramam 10 2 2 1 2 4 1 2 7 2 2 2 2 2 2

351 808 1 1 78 Mannurpet 7 2 2 1 2 3 1 2 7 2 2 2 2 2 2

352 809 2 1 72 Anna nagar 8 3 1 1 2 3 1 2 7 2 2 2 2 2 2

353 810 2 1 71 Kolathur 6 2 2 1 2 1 1 2 7 2 2 2 2 2 2

354 811 1 1 75 Kolathur 6 2 2 1 2 1 1 2 7 2 2 2 2 2 2

355 812 1 1 93 Gummidipondi 16 2 2 1 2 3 1 2 7 2 2 2 2 2 2

356 813 4 1 86 Trichy 16 1 1 1 2 3 1 2 7 2 2 2 2 2 1

357 814 1 1 63 Mayilladurai 16 2 2 1 1 2 1 2 7 2 2 2 2 2 2

358 815 1 1 70 Vadapalani 10 2 2 1 1 3 1 2 7 2 2 2 2 2 2

359 816 1 1 97 Puzhal 3 2 2 1 1 2 1 2 7 2 2 2 2 2 2

360 891 1 1 77 Sowcarpet 8 3 2 1 1 3 1 2 7 2 2 2 2 2 2

361 892 2 1 66 Teachers colony 13 2 2 1 1 3 1 2 7 2 2 2 2 2 2

362 893 2 1 67 Puzhal 3 1 1 1 2 3 1 2 7 2 2 2 2 2 2

363 894 1 1 63 Choolaimedu 8 2 2 1 1 2 1 2 7 2 2 2 2 2 2

364 895 1 1 85 Kolathur 6 2 2 1 1 3 1 2 7 2 2 2 2 2 2

365 896 1 1 70 Egmore 8 1 1 1 1 1 1 2 7 2 2 2 2 2 2

366 897 3 1 90 Anna nagar 8 1 2 1 2 3 1 2 7 2 2 2 2 2 2

367 898 1 1 60 vadapalani 8 2 2 1 1 2 1 2 7 2 2 2 2 2 2

368 899 2 1 90 Egmore 8 1 2 1 1 2 1 2 7 2 2 2 2 2 2

369 900 1 1 67 Purasaivakkam 8 2 2 1 1 1 1 2 7 2 2 2 2 2 2

370 901 1 1 118 ICF,konnur 7 2 2 1 2 2 1 2 7 2 2 2 2 2 2

371 902 1 1 83 Ennore 1 2 2 1 1 3 1 2 7 2 2 2 2 2 2

372 903 1 1 90 Little mount 13 2 2 1 1 1 1 2 7 2 2 2 2 2 2

373 904 1 1 77 Kolathur 6 2 2 1 1 2 1 2 7 2 2 2 2 2 2

RT PCTR HCV

Age Gender Blood group Rh type No.Donation Seropositive detetion No of copies( IU/ml) GENOTYPE 25 Male O Negative First time HCVAg-Ab ELISA HCV RNA 2,84.000 GENOTYPE 1b

RT PCR HBV

Age Gender Blood group Rh type No.Donation Seropositive detetion 27 Male O positive Repeat HBsAg ELISA HBV DNA 21 Male A1 positive Repeat HBsAg ELISA HBV DNA 27 Male A1 positive Repeat HBsAg ELISA HBV DNA

Age group in years Code Month Code 18-20 1 Aug-18 8 21-30 2 Sep-18 9 31-40 3 Oct-18 10 41-50 4 Nov-18 11 >50 5 Dec-18 12 Jan-19 1 Gender Code Feb-19 2 Male 1 Mar-19 3 Female 2 Apr-19 4 May-19 5 Occupation Code Jun-19 6 working(employed) 1 Jul-19 7 Student 2 others 3 3rd generation ELISA anti HIV I & II Code Positive 1 Negative 2 Socio economic status Code Indeterminate 3 High 1 Middle 2 anti HCV Code Low 3 Positive 1 Negative 2 Blood Group Code Indeterminate 3 A1 1 B 2 HBsAg Code O 3 Positive 1 A1B 4 Negative 2 A2B 5 Indeterminate 3 A2 6 4th generation ELISA Rh Typing Code anti-HIV I & II and P24 Ag Code Positive 1 Reactive 1 Negative 2 Nonreactive 2 Indeterminate 3

Repeat test code anti-HCV and core Ag Code Reactive 1 Reactive 1 Non Reactive 2 Nonreactive 2 Indeterminate 3 Indeterminate 3

Type of Donor Code HBsAg and Variants Code Voluntary 1 Reactive 1 Replacement 2 Nonreactive 2 Indeterminate 3

Zone Code Thiruvotriyur 1 Manali 2 Madhavaram 3 Tondiarpet 4 Royapuram 5 Thiru-vi-ka Nagar 6 Ambattur 7 Anna Nagar 8 Teynampet 9 Kodambakkam 10 valasaravakkam 11 Alanthur 12 Adyar 13 Perungudi 14 Sholinganallur 15 outside Chennai 16