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Molecular Techniques for Blood and Blood Product Screening

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Abstract

Blood product safety is a high priority for manufacturing industries, hospitals, and regulatory agencies. An important step in ensuring safety is the screening of donated blood for infectious diseases. Molecular technologies for screening infectious diseases have improved remarkably over the years. Molecular biological assay significantly reduced the risk of transfusion-transmitted infections. Unlike previous methods, molecular technologies for screening infectious diseases are specific, efficient, easy to use, and economical. A new era in molecular biology is coming to the field of blood safety.

Introduction

The Food and Drug Administration (FDA) is responsible for ensuring the safety of more than 15 million units of blood and blood components donated each year in the United States. “Blood banking has become a manufacturing industry, an industry that must conform to high standards and quality control requirements comparable to those of pharmaceutical companies or other regulated industries,” said David A. Kessler, M.D., former FDA commissioner [1]. Screening donated blood for infectious diseases that can be transmitted through blood transfusion is a very important step in ensuring safety. The United States has the safest blood supply in the world [1], and the FDA is striving to keep it safe by decreasing the risk of infectious disease transmission. The regulatory agency is continuously updating its requirements and standards for collecting and processing blood. As mentioned earlier, an important step in ensuring safety is the screening of donated blood for infectious diseases [2, 3]. In the United States, tests for infectious diseases are routinely conducted on each unit of donated blood, and these tests are designed to comply with regulatory requirements (Table 1). The field of clinical microbiology and virology is now focusing on molecular technology. Currently, nucleic acid testing techniques have been developed to screen blood and plasma products for evidence of very recent viral infections that could be missed by conventional serologic tests [2]. It is time for all blood safety procedures to include molecular detection techniques [3]. This approach can significantly aid in blood safety to reduce the risk of transmission of serious disease by transfusion. This chapter will review and update the current antigen/antibody-based technology, molecular biological technology, and published regulatory policy data for blood safety.
Table 1

Licensed/approved clinical assays for infectious agents

Trade name

Infectious agent

Format

Specimen

Use

Manufacturer

Approval date

STN

HBsAg assays (detect hepatitis B surface antigen)

Abbott PRISM HBsAg; Abbott PRISM HBsAg Confirmatory

HBV

ChLIA

ChLIA-specific antibody neutralization

Serum/plasma/cadaveric serum

Donor screening: qualitative detection of HBsAgConfirmatory: to confirm the presence of HBsAg in samples found to be repeatedly reactive by the Abbott PRISM HBsAg assay

Abbott LaboratoriesAbbott Park, ILUS License 0043

7/18/2006

BL103766

Genetic Systems HBsAg EIA 3.0;Genetic Systems HBsAg Confirmatory Assay 3.0

HBV

EIA

Serum/plasma/cadaveric serum

In vitro diagnosticDonor screening: qualitative detection of HBsAg Confirmatory: to confirm the presence of HBsAg in reactive specimens

Bio-Rad LaboratoriesRedmond, WAUS License 1109

1/23/2003

BL103590

Anti-HBc assays (detect antibodies to hepatitis B virus core antigen)

ORTHO HBc ELISA Test System

HBV

ELISA

Serum/plasma

In vitro diagnostic, Donor screening: qualitative detection of total antibody to hepatitis B core antigen

Ortho-Clinical Diagnostics, IncRaritan, NJ

4/23/1998

BL103062

Abbott PRISM HBcore

HBV

ChLIA

Serum/plasma

Donor screening, qualitative detection of total antibody to hepatitis B core antigen

Abbott LaboratoriesAbbott Park, ILUS License 0043

10/13/2005

BL103785

HBV nucleic acid assays (detect hepatitis B virus DNA); see also multiplex assays

UltraQual™ HBV PCR Assay

HBV

PCR

Plasma

Source plasma donor screening: qualitative detection of HBV DNA. For in-house use only at NGI

National Genetics Institute Los Angeles, CA US License 1582

9/1/2011

BL125193

COBAS AmpliScreen HBV Test

HBV

PCR

Plasma/cadaveric serum or plasma

Donor screening: qualitative detection of HBV DNA

Roche Molecular Systems, Inc.Pleasanton, CAUS License1636

4/21/2005

BL125090

Anti-HCV assays (detect antibodies to hepatitis C virus encoded antigen)

Abbott PRISM HCV

HCV

ChLIA

Serum /plasma/cadaveric serum

Donor screening: qualitative detection of antibodies to hepatitis C virus

Abbott LaboratoriesAbbott Park, IL US License 0043

7/11/2007

BL103762

Ortho HCV Version 3.0 ELISA Test System

HCV

EIA

Serum/plasma

Donor screening: qualitative detection of antibodies to hepatitis C virus

Ortho-Clinical Diagnostics, IncRaritan, NJUS License 1236

4/23/19982/18/2009

BL103065

HCV nucleic acid assays (detect hepatitis C virus RNA); see also multiplex assays

Hepatitis C Virus RT PCR Assay

HCV

PCR

Plasma

Source plasma donor screening: qualitative detection of HCV RNA. For in-house use only by Baxter Healthcare International

BioLife Plasma Services, L.P.Deerfield, ILUS License 1640

2/9/2007

BL125101

UltraQual HCV RT-PCR Assay

HCV

PCR

Plasma

Source plasma donor screening: qualitative detection of HCV RNA. For in-house use only at NGI

National Genetics InstituteLos Angeles, CAUS License 1582

9/18/2001

BL103868

COBAS AmpliScreen HCV Test, ver 2.0

HCV

PCR

Plasma/cadaveric serum or plasma

Donor screening: qualitative detection of HCV RNA.Donor supplemental: to confirm HCV infection for specimens that are repeatedly reactive on a licensed donor screening test for antibodies to HCV and reactive on the COBAS® AmpliScreen HCV Test, v2.0

Roche Molecular Systems, IncPleasanton, CAUS License 1636

12/3/2002

BL125045

Anti-HIV-1 assays (detect antibodies to human immunodeficiency virus type 1)

Fluorognost HIV-1 IFA

HIV-1

IFA

Serum/plasmaDried blood spot

Diagnostic and donor supplemental: qualitative detection of antibodies to HIV-1 for use as an additional, more specific test in specimens found to be repeatedly reactive by screening proceduresDonor screening: only in special cases

Sanochemia Pharmazeutika AGVienna, AustriaUS License 1631

2/5/19925/14/1996

BL103288BL103651

Cambridge Biotech HIV-1 Western Blot Kit

HIV-1

WB

Serum /plasma

Diagnostic and donor supplemental: qualitative detection of antibodies to HIV-1 for use as an additional, more specific test in specimens found to be repeatedly reactive by screening procedures

Maxim Biomedical, Inc.Rockville, MDUS License 1741

5/28/1998

BL103843

GS HIV-1 western blot

HIV-1

WB

Serum/plasmaDried blood spot

Diagnostic and donor supplemental: qualitative detection of antibodies to HIV-1 for use as an additional, more specific test in specimens found to be repeatedly reactive by screening procedures

Bio-Rad LaboratoriesRedmond, WAUS License 1109

11/13/1998

BL103655

Avioq HIV-1 Microelisa System

HIV-1

EIA

Serum, plasma, dried blood spot, oral fluid

In vitro diagnostic: qualitative detection of antibodies to HIV-1

Avioq Inc.,Rockville, MD

9/21/2009

BP090022

Maxim (Calypte) HIV-1 Urine EIA

HIV-1

EIA

Urine

In vitro diagnostic: qualitative detection of antibodies to HIV-1

Maxim Biomedical, Inc.Rockville, MDUS License 1741

1/12/2001

BP000009

INSTI HIV-1/HIV-2 Antibody Test

HIV-1 and HIV-2

Rapid immunoassay

Plasma /whole blood (venipuncture and finger stick)

In vitro diagnostic: qualitative detection of antibodies to HIV-1

bioLytical Laboratories Inc.British Columbia, Canada

1/28/2015

BP090032/7

Reveal Rapid HIV-1 Antibody Test

HIV-1

Rapid immunoassay

Serum/plasma

In vitro diagnostic: qualitative detection of antibodies to HIV-1; point-of-care test

MedMira Laboratories, Inc.Halifax, Nova ScotiaCanada

4/16/2003

BP000023

OraSure HIV-1 Western Blot Kit

HIV-1

WB

Oral fluid

Diagnostic supplemental: qualitative detection of antibodies to HIV-1 for use as an additional, more specific test in oral fluid specimens found to be repeatedly reactive by the Oral Fluid Vironostika HIV-l Microelisa System

OraSure TechnologiesBethlehem, PA

6/3/1996

BP950004

Cambridge Biotech HIV-1 Urine Western Blot Kit

HIV-1

WB

Urine

Diagnostic supplemental: qualitative detection of antibodies to HIV-1 for use as an additional, more specific test in urine specimens found to be repeatedly reactive by Maxim HIV-1 Urine EIA

Maxim Biomedical, Inc.Rockville, MDUS License 1741

6/21/2001

BP010009

HIV-1 nucleic acid assays (detect human immunodeficiency virus type 1 RNA); see also multiplex assays

Human Immunodeficiency Virus, Type 1 RT PCR Assay

HIV-1

Qualitative PCR

Plasma

Source plasma donor screening: qualitative detection of HIV-1 RNA. For in-house use only by Baxter Healthcare International

BioLife Plasma Services, L.P.Deerfield, ILUS License 1640

1/31/2007

BL125100

UltraQual HIV-1 RT-PCR Assay

HIV-1

Qualitative PCR

Plasma

Source plasma donor screening: qualitative detection of HIV-1 RNA. For in-house use only at NGI

>National Genetics InstituteLos Angeles, CAUS License 1582

9/18/2001

BL103902

COBAS AmpliScreen HIV-1 Test, ver 1.5

HIV-1

Qualitative PCR

Plasma/cadaveric serum or plasma

Donor screening: qualitative detection of HIV-1 RNA

Roche Molecular Systems, Inc.Pleasanton, CAUS License 1636

12/20/2002

BL125059

APTIMA HIV-1 RNA Qualitative Assay

HIV-1

TMA

Plasma/serum

In vitro diagnostic: qualitative detection of HIV-1 RNA

Gen-Probe, Inc.,San Diego, CAUS License 1592

10/4/2006

BL103966

COBAS HIV-1

HIV-1

Quantitative PCR

Plasma

Patient monitoring: quantitation of HIV-1 RNA in plasma of HIV-1 infected individuals

Roche Molecular Systems, Inc.Pleasanton, CAUS license 1636

12/18/2015

BP150262

Abbott RealTime HIV-1

HIV-1

Quantitative PCR

Plasma

Patient monitoring: quantitation of HIV-1 RNA in plasma of HIV-1 infected individuals

ABBOTT Molecular, Inc., Des Plaines, IL

5/11/2007

BP060002

Amplicor HIV-1 Monitor Test

HIV-1

Quantitative PCR

Plasma

Patient monitoring: quantitation of HIV-1 RNA in plasma of HIV-1 infected individuals.

Roche Molecular Systems, Inc.Pleasanton, CAUS License 1636

3/2/1999

BP950005

COBAS AmpliPrep/COBAS TaqMan HIV-1 Test

HIV-1

Quantitative PCR

Plasma

Patient monitoring: quantitation of HIV-1 RNA in plasma of HIV-1 infected individuals

Roche Molecular Systems, Inc.Pleasanton, CAUS License 1636

5/11/2007

BP050069

Versant HIV-1 RNA 3.0 (bDNA)

HIV-1

Quantitative PCR

Plasma

Patient monitoring: quantification of HIV-1 RNA in plasma of HIV-1 infected individuals

Siemens Healthcare Diagnostics, Inc.

9/11/2002

BP000028

ViroSeq HIV-1 Genotyping System

HIV-1

Genotyping

Plasma

Patient monitoring: for detecting HIV genomic mutations (in the protease and part of the reverse transcriptase regions of HIV) that confer resistance to specific types of antiretroviral drugs, as an aid in monitoring and treating HIV infection

Celera DiagnosticsAlameda, CA

6/11/20037/10/2008

BK030033,BK080026

Trugene HIV-1 Genotyping Kit and Open Gene DNA Sequencing System

HIV-1

Genotyping

Plasma

Patient monitoring: for detecting HIV genomic mutations (in the protease and part of the reverse transcriptase regions of HIV) that confer resistance to specific types of antiretroviral drugs, as an aid in monitoring and treating HIV infection

Siemens Healthcare Diagnostics, Inc.

4/24/20023/27/2012

BK020005, BK120013

Anti-HIV specimen collection devices, testing services, and home test kits

Home Access HIV-1 Test System

HIV-1

Dried blood spot collection device

Dried blood spot

In vitro diagnostic: self-use by people who wish to obtain anonymous HIV testing

Home Access Health Corp.,Hoffman Estates, IL

7/22/1996

BP950002

OraSure HIV-1 Oral Specimen Collection Device

HIV-1

Oral specimen collection device

Oral fluid

For use with HIV diagnostic assays that have been approved for use with this device

OraSure TechnologiesBethlehem, PA

12/23/1994

BP910001

OraQuick In-Home HIV Test

HIV-1, HIV-2

Immunoassay

Oral fluid

Over-the-counter (OTC) diagnostic home-use test. A positive result is preliminary, and follow-up confirmatory testing is needed

OraSure TechnologiesBethlehem, PA

07/03/2012

BP120001

Anti-HIV-2 assays (detect antibodies to human immunodeficiency virus type 2)

Genetic Systems HIV-2 EIA

HIV-2

EIA

Serum/plasma

In vitro diagnostic, Donor screening: qualitative detection of antibodies to HIV-2

Bio-Rad LaboratoriesRedmond, WAUS License 1109

4/25/1990

BL103227

Anti-HIV-1/2 assays (detect antibodies to human immunodeficiency virus types 1 and 2)

Abbott PRISM HIV O Plus assay

HIV-1, HIV-2

ChLIA

Plasma/serum/cadaveric serum

In vitro diagnosticDonor screening: qualitative detection of antibodies to HIV-1 groups M and O and/or antibodies to HIV-2

Abbott LaboratoriesAbbott Park, ILUS License 0043

9/18/2009

BL125318

Geenius HIV 1/2 Supplemental Assay

HIV-1, HIV-2

HIV detection test

Blood

Used as specific test to confirm the presence of antibodies of HIV-1 and HIV-2 for the detection of specimens to be repeatedly reactive by screening procedures

Bio-Rad Laboratories, IncRedmond, WAUS License 1109

10/24/2014

BP140120

Genetic Systems HIV-1/HIV-2 Plus O EIA

HIV-1, HIV-2

EIA

Serum/plasma/cadaveric serum

In vitro diagnostic Donor screening: qualitative detection of antibodies to HIV-1 (groups M and O) and/or HIV-2

Bio-Rad LaboratoriesRedmond, WAUS License 1109

8/5/2003

BL125030

ADVIA Centaur HIV 1/O/2 Rapid Test

HIV-1, HIV-2

Microparticle chemi-luminometric immunoassay

Plasma/serum

In vitro diagnostic: qualitative determination of antibodies to HIV-1, including group O, and/or HIV-2

Siemens Healthcare Diagnostics, Inc.

5/18/2006

BP050030

VITROS HIV-1/HIV-2 Reagent Pack and Calibrator

HIV-1, HIV-2

Immunometric

Plasma/serum

In vitro diagnostic: qualitative detection of antibodies to HIV-1 and/or HIV-2

Ortho-Clinical Diagnostics, IncRaritan, NJUS License 1236

3/27/2008

BP050051

Multispot HIV-1/HIV-2 Rapid Test

HIV-1, HIV-2

Rapid immunoassay

Plasma/serum

In vitro diagnostic: qualitative detection of antibodies to HIV-1 and/or HIV-2

Bio-Rad LaboratoriesRedmond, WAUS License 1109

11/12/2004

BP040046

SURE CHECK HIV 1/2 ASSAY

HIV-1, HIV-2

Rapid immunoassay

Finger stick and venous whole blood, serum, plasma

In vitro diagnostic: qualitative detection of antibodies to HIV-1 and/or HIV-2; point-of-care test

Chembio Diagnostic Systems, Inc.Medford, NY

5/25/2006

BP050009

HIV 1/2 STAT-PAK ASSAY

HIV-1, HIV-2

Rapid immunoassay

Finger stick and venous whole blood, serum, plasma

In vitro diagnostic: qualitative detection of antibodies to HIV-1 and/or HIV-2; point-of-care test

Chembio Diagnostic Systems, Inc.Medford, NY

5/25/2006

BP050010

OraQuick ADVANCE Rapid HIV-1/2 Antibody Test

HIV-1, HIV-2

Rapid immunoassay

Oral fluid, plasma, whole blood (venipuncture and finger stick)

In vitro diagnostic: qualitative detection of antibodies to HIV-1 and/or HIV-2; point-of-care test

OraSure TechnologiesBethlehem, PA

11/7/20026/22/2004

BP010047

Chembio DPP® HIV 1/2 Assay

HIV-1, HIV-2

Rapid immuno-chromatographic assay

Oral fluid, serum, plasma, Whole blood (venipuncture, finger stick)

In vitro diagnostic: qualitative detection of antibodies to HIV-1 and/or HIV-2; point-of-care test

Chembio Diagnostic Systems, Inc.Medford, NY

12/19/2012

BP120032

Uni-Gold™ Recombigen® HIV-1/2

HIV-1, HIV-2

Rapid EIA

Serum, plasma, Whole blood (venipuncture, finger stick)

In vitro diagnostic: qualitative detection of antibodies to HIV-1 and/or HIV-2; point-of-care test

Trinity BiotechJamestown, NY

12/23/200302/04/2013

BP030025

Anti-HIV-1/2 and HIV-1 antigen combo assays (detect HIV-1 antigen and antibodies to HIV types 1 and 2)

BioPlex 2200 HIV Ag-Ab Assay

HIV-1 and HIV-2

Multiplex flow immunoassay

Serum, plasma

In vitro diagnostic: simultaneous qualitative detection and differentiation of HIV p24 antigen and antibodies to HIV-1 (group M and group O) and/or HIV-2

Bio-Rad LaboratoriesRedmond, WAUS License 1109

7/23/2015

BP140111

ADVIA Centaur HIV Ag/Ab Combo (CHIV) Assay

HIV-1, HIV-2

Microparticle chemi-luminometric immunoassay

Serum

In vitro diagnostic: simultaneous qualitative detection and differentiation of HIV p24 antigen and antibodies to HIV-1 (group M and group O) and/or HIV-2

Siemens Healthcare Diagnostics, Inc.

6/8/2015

BP140103

ARCHITECT HIV Ag/Ab Combo

HIV-1, HIV-2

CMIA

Plasma/serum

In vitro diagnostic: simultaneous qualitative detection of HIV p24 antigen and antibodies to HIV-1 (group M and group O) and/or HIV-2

Abbott LaboratoriesAbbott Park, ILUS License 0043

6/18/2010

BP090080

GS HIV Ag/Ab Combo EIA

HIV-1, HIV-2

EIA

Plasma/serum

In vitro diagnostic: simultaneous qualitative detection of HIV p24 antigen and antibodies to HIV-1 (group M and group O) and/or HIV-2

Bio-Rad LaboratoriesRedmond, WAUS License 1109

7/22/2011

BP100064

Alere DetermineTMHIV-1/2 Ag/Ab Combo

HIV-1, HIV-2

Immunoassay

Serum, plasma, Whole blood (venipuncture, finger stick)

In vitro diagnostic: simultaneous qualitative detection of HIV p24 antigen and antibodies to HIV-1 and/or HIV-2; point-of-care test

Alere Scarborough, Inc.Scarborough, ME

8/8/2013

BP120037

Anti-HTLV-I/II Assays (detect antibodies to Human T-Lymphotropic Virus types I and II)

Avioq HTLV-I/II Microelisa System

HTLV-I, HTLV-II

EIA lysate

Serum/plasma

In vitro diagnostic, Donor screening: qualitative detection of antibodies to HTLV-I and HTLV-II

Avioq, Inc. Research Triangle Park, NC 27709US License 1856

03/26/2012

BL125394

Abbott PRISM HTLV-I/HTLV-II

HTLV-I, HTLV-II

ChLIA

Serum/plasma

Donor screening: qualitative detection of antibodies to HTLV-I and HTLV-II

Abbott LaboratoriesAbbott Park, ILUS License 0043

1/16/2008

BL103761

MP Diagnostic HTLV Blot 2.4

HTLV-I, HTLV-II

WB

Serum/plasma

Donor supplemental: qualitative detection and differentiation of antibodies to HTLV-I and HTLV-II for use as an additional, more specific test in specimens found to be repeatedly reactive by screening procedures

MP Biomedicals Asia Pacific PTE. LTD

12/11/2014

BL125475

Anti-T. pallidum assays (detect antibodies to Treponema pallidum) and other serological tests for syphilis

CAPTIATM Syphilis (T. Pallidum)-G

T. Pallidum

EIA

Serum, plasma

Donor screening: qualitative detection of IgG antibodies to Treponema pallidum

Trinity BiotechJamestown, NY

01/24/2002

K014233

ASI TPHA Test

T. Pallidum

Micro-hemagglutination

Serum

Donor screening: qualitative detection of IgG and IgM antibodies to Treponema pallidum

Arlington Scientific, IncSpringville, UT

1/30/2003

BK020031

Olympus PK TP System

T. Pallidum

Micro-hemagglutination

Serum, plasma

Donor screening: qualitative detection of IgG and IgM antibodies to Treponema pallidum

Fujirebio Diagnostics Inc..Malvern, PA

2/21/2003

BK030007

TPHA Screen

T. Pallidum

Hemagg-lutination

Serum, plasma

Donor screening: qualitative detection of IgG and IgM antibodies to Treponema pallidum

Immucor, Inc.Norcross, GA

10/24/2012

BK120021

ASiManager-AT™

T. Pallidum

Rapid plasma reagin (RPR) test

Serum, plasma

Donor screening: qualitative detection of reagin antibodies

Arlington Scientific, Inc. Springville, UT.

2/24/2014

BK 130001

Anti-T. cruzi assays (detect antibodies to Trypanosoma cruzi)

Abbott ESA Chagas

T. Cruzi

Multistep enzyme strip immunoassay

Serum, plasma

An additional, more specific test on human serum or plasma specimens found to be repeatedly reactive using a licensed screening test for antibodies to T. cruzi

Abbott LaboratoriesAbbott Park, ILUS License 0043

11/18/2011

125,361/23

Abbott PRISM Chagas

T. Cruzi

EIA

Serum/plasma

Donor screening: qualitative detection of antibodies to T. cruzi

Abbott LaboratoriesAbbott Park, ILUS License 0043

4/30/2010

BL125361

ORTHO T. cruzi ELISA Test System

T. Cruzi

EIA

Serum/plasma/cadaveric

Donor screening: qualitative detection of antibodies to T. cruzi

Ortho-Clinical Diagnostics, Inc.Raritan, NJUS License 1236

12/13/2006

BL125161

WNV nucleic acid assays (detect West Nile virus RNA)

Procleix West Nile Virus (WNV) Assay

WNV

Nucleic acid test (TMA)

Plasma/cadaveric plasma or serum

Donor screening: qualitative detection of West Nile virus (WNV) RNA.

Gen-Probe, Inc.,San Diego, CAUS License 1592

12/1/2005

BL125121

COBAS TaqScreen West Nile Virus Test

WNV

PCR

Plasma/cadaveric plasma or serum

Donor screening: qualitative detection of West Nile virus (WNV) RNA

Roche Molecular Systems, Inc.Pleasanton, CAUS License 1636

8/28/2007

BL125245

Anti-CMV assays (detect antibodies to Cytomegalovirus)

Capture-CMV

CMV

Solid-phase red cell adherence

Serum, plasma

Donor screening: qualitative detection of IgG and IgM antibodies to CMV

Immucor, Inc.Norcross, GA

12/22/1995

BK950029

Olympus PK CMV-PA System

CMV

Passive particle agglutination

Serum, plasma

Donor screening: qualitative detection of IgG and IgM antibodies to CMV

Fujirebio Diagnostics, Inc.Malvern, PA

9/20/2007

BK070030

Multiplex assays

COBAS TaqScreen MPX Test

HBV, HCV, HIV-1, HIV-2

PCR

Plasma/cadaveric plasma or serum

Donor screening: simultaneous qualitative detection of HBV DNA, HIV-1 group M and group O RNA, HIV-2 RNA, and HCV RNA

Roche Molecular Systems, Inc.Pleasanton, CAUS License 1636

12/30/2008

BL125255

COBAS TaqScreen MPX Test version 2.0

HBV, HCV, HIV-1, HIV-2

PCR

Plasma

Donor screening: simultaneous qualitative detection of HBV DNA, HIV-1 group M and group O RNA, HIV-2 RNA, and HCV RNA

Roche Molecular Systems, Inc.Pleasanton, CAUS License 1636

12/19/2014

BL 125459

Procleix Ultrio Assay

HBV, HCV, HIV-1

Nucleic acid test (TMA

Plasma/serum/cadaveric plasma or serum

Donor screening: simultaneous qualitative detection of HBV DNA, HCV RNA, and HIV-1 RNA

Gen-Probe, Inc.,San Diego, CAUS License 1592

10/3/2006

BL125113

Procleix Ultrio Plus Assay

HBV, HCV, HIV-1

Nucleic acid test (TMA)

Plasma/serum/cadaveric plasma or serum

Donor screening: simultaneous qualitative detection of HBV DNA, HCV RNA, and HIV-1 RNA

Gen-Probe, Inc.,San Diego, CAUS License 1592

5/25/2012

BL125113

Source: Center for Biologics Evaluation and Research, US Food and Drug Administration

Limitations for Current Technologies Used in Blood Safety

Direct detection of viral antigens and virus-specific antibodies has been a common tool for the diagnosis of virus infections in the past 40 years. There are some limitations. For direct detection of virus antigens, shortly after virus infection, only a few viruses release antigens in amounts sufficiently detectable in the body by an antibody-mediated assay. For indirect virus detection by virus-specific antibodies [e.g., an immunofluorescence assay or enzyme immunoassay (EIA), etc.], there is a problem in that shortly after infection by a pathogenic virus, and there is a window period in which antibody generation is insufficient for detection [4]. To reduce this window period of low detection, direct nucleic acid tests are needed.

Application of Advanced Molecular Techniques in Blood Safety Applications

Through the application of molecular biology, biological and biochemical analyses have been revolutionized, and nucleic acid, gene-based techniques have been developed to screen blood and plasma donations for evidence of very recent and earlier viral infections that might otherwise be missed by conventional serologic testing. The nucleic acid tests can also provide evidence for genetic variation in viruses. Molecular methods include the use of nucleic acid probes as well as amplification-based and DNA sequence-based techniques. An increasing number of molecular diagnostic methods are now available commercially [2]. In comparison to classical methods, molecular biological methods are superior in terms of rapidness, specificity, and sensitivity. The current nucleic acid detection methods in the field may be grouped into two major classes: amplifying techniques such as PCR and non-amplifying techniques such as Southern blot hybridization. Amplifying techniques are more sensitive than non-amplifying techniques. There are two different types of amplifying methods [5], target amplification methods and signal amplification methods. Target amplifying techniques include PCR, nucleic acid sequence-based amplification (NASBA) [6, 7], self-sustaining sequence amplification (3SR), transcription-based amplification (TAS), transcription-mediated amplification (TMA), strand displacement amplification (SDA), and ligase chain reaction (LCR). Signal amplification methods include branched DNA signal amplification (bDNA) [8], cleavage-based signal amplification (cycling probe technologies and invader assay), Qß replicase, hybrid capture, cycling probe technologies (CPT), and rolling-circle amplification (RCA) [9]. To further insure the safety of blood products, it is of importance to further improve these and other types of nucleic acid testing [2, 3, 4, 5].

Major Different Generations of Nucleic Acid Detection Techniques

Southern Blot Hybridization (1970s)

Southern blotting [10] was named after Edward M. Southern who developed this procedure at Edinburgh University in the 1970s. This technique is used to detect specific sequences within mixtures of DNA, which is size fractionated by gel electrophoresis and then transferred by capillary action to a suitable membrane. After blocking of non-specific binding sites, the nitrocellulose replica of the original gel electrophoresis experiment is then allowed to hybridize with an oligonucleotide probe representing the specific DNA sequence of interest. Should specific DNA be present on the blot, it will combine with the labeled probe and be detectable. By coelectrophoresing DNA fragments of known molecular weight, the size(s) of the hybridizing band(s) can then be determined. Southern blotting hybridization technology is one of the major tools that have already helped clinical staffs worldwide interpret genomic information. Other competing methodologies include in situ hybridization and solution hybridization. Important clinical examples of the use of this technology are DNA fingerprinting and the ability to detect DNA gene rearrangements.

Polymerase Chain Reaction (1980s)

In 1983, Dr. Kary Mullis at Cetus Corporation conceived of polymerase chain reaction [11]. There is not a single technique that has had a greater impact on the practice of molecular biology than PCR. With this technique, we can detect infectious disease agents at an extremely low level. It is based on the ability of sense and antisense DNA primers to hybridize to a DNA of interest. Following extension from the primers on the DNA template by DNA polymerase, the reaction is heat denatured and allowed to anneal with the primers once again. Another round of extension leads to a multiplicative increase in DNA products. Therefore, a minute amount of DNA can be efficiently amplified in an exponential fashion to result in larger amounts of DNA that are more easily manipulated. By including critical controls, the technique can be made quantitative. The current level of the sensitivity and detection limit is as low as 10–50 copies per ml blood in HIV testing [1, 12, 13]. Important clinical examples of the use of PCR are detection of HIV and HCV [14, 15, 16]. PCR techniques have evolved into different branches. Some of them are now widely in use for virus detection in clinical diagnostics. These are real-time PCR by TaqMan (Roche), LightCycler (Roche), Smartcycler (Cepheid), in situ PCR, nested PCR, nested real-time PCR [17], broad-range PCR, multiplex PCR, RT-PCR, arbitrarily primer PCR, long PCR, and quantitative PCR. Real-time sequence technology will be coming soon for more detailed detection. In the past, identification of viral serotypes was restricted to investigative methods using antibody detection and restriction fragment length polymorphism (RFLP). With real-time sequence technology, we will be able to detect a virus early as well as to obtain the viral sequence.

Microarrays (1990s)

Microarrays were developed at Stanford University by Schena and co-workers in the early 1990s [18]. For medical applications, a microarray analysis offers a very accurate screening technology. It allows hundreds or thousands of nucleic acid hybridization reaction to be performed on a solid substrate. It promises to be a fast and accurate diagnostic tool in the field of clinical microbiology and virology. Applied to infection safety for blood and blood products, it will be able to screen for the presence of viral pathogens by matching genetic sequences. Compared with existing technologies, it allows for a wider variety of specific tests to be carried out simultaneously to determine the quality of the blood and will provide consumers with extra safety. With the use of molecular biology protocols, the microarray will permit the detection of lower concentrations of microorganisms in the blood and the accurate identification of many types of pathogenic contaminants. In the near future, progress can be expected in the application of microarray technology for screening of donated blood for infectious agents. It can provide vast information about the identity of blood-borne pathogens as well as their gene expression profiles [19].

Screening of Donor Blood for Infectious Agents

To ensure a safe blood supply for those who may need a transfusion, an important step in ensuring safety is the screening of donated blood for infectious agents [20]. After donation, each unit of donated blood undergoes a series of tests for blood-borne agents such as human immunodeficiency virus (HIV)-1, HIV-2, hepatitis B virus (HBV), hepatitis C virus (HCV), human T-cell lymphotropic virus (HTLV)-1 and HTLV-II, West Nile virus (WNV), Treponema pallidum (syphilis), Trypanosoma cruzi (T. cruzi), and cytomegalovirus.

Confirmatory Testing of Donor Blood for Infectious Agents

All of the above tests are referred to as screening tests and are designed to detect as many infectious agents as possible. Because these tests are so sensitive, some donors may have a false-positive result, even when the donor has never been exposed to the particular infection. In order to sort out true infections from such false-positive test results, screening tests that are reactive may be followed up with more specific tests called confirmatory tests. Thus, confirmatory tests help determine whether a donor is truly infected. If any one of these tests fails, affected blood products are considered unsuitable for transfusion [20].

Application of Nucleic Acid Testing for Infectious Agents

Nucleic acid testing (NAT) employs testing technology that directly detects the genomes of viruses. Because NAT detects a virus’s genetic material instead of waiting for the body’s response, the formation of antibodies, as with many current tests, it offers the opportunity to reduce the window period during which an infecting agent is undetectable by traditional tests [21], thus further improving blood safety. Nucleic acid testing is becoming the gold standard because of greater sensitivity compared to antibody tests [2].

Since 1999, NAT has been approved by the FDA and used to detect HIV-1 and HCV; this technology currently is under investigation for detecting other infectious disease agents. We know that for many viral infections, viral RNA appears very early in the infection, in 1 to 2 weeks, but the antibody doesn’t appear until 10–12 weeks, e.g., HIV and HCV [21]. In order to virtually prevent infection by all the transfusion-associated viruses, we need to detect the viruses in their window period, and a NAT or gene-based testing method is needed. NAT also provides an opportunity for the viral, e.g., HIV or HCV, infected donor to seek early treatment. On the other hand, NAT is not only a sensitive method but also a rapid method, which is suitable for a blood bank laboratory because the turnaround time for maintaining blood donations is extremely critical.

Hepatitis B Virus

The hepatitis B virus (HBV) is a highly infectious and often non-symptomatic virus that is transmitted primarily through blood and blood-derived fluids and is a leading cause of liver infection worldwide [22]. The World Health Organization (WHO) estimates that 2 billion people worldwide have been infected with HBV and 350,000,000 people are chronically infected. Chronic infection results in a high risk for liver cancer and cirrhosis of the liver, which cause about 1000,000 deaths each year. Each year up to 200,000 people become newly infected in the United States alone. Since screening for HBV began in 1969, the rate of infection through blood transfusions has greatly decreased. However, as of 2000, HBV is still transmitted through blood transfusions in 1 out of 137,000 units of blood. One reason for this is that currently available blood screening technologies detect core antibodies or surface antigens, which appear up to 8 weeks after infection. Serologic tests for hepatitis B virus include hepatitis B surface antigen (HBsAg) and hepatitis B core antibody (HBcAb).

Hepatitis B Surface Antigen

HBV, which mainly infects the liver, has an inner core and an outer envelope (the surface). The HBsAg test detects the outer envelope, identifying an individual infected with the hepatitis B virus. This virus can cause inflammation of the liver, and in the earliest stage of the disease, infected people may feel ill or even have yellow discoloration of the skin or eyes, a condition known as jaundice. Fortunately, most patients recover completely and test negative for HBsAg within a few months after the illness. A small percentage of people become chronic carriers of the virus, and in these cases, the test may remain positive for years. Chronically infected people can develop severe liver disease as time passes and need to be followed carefully by an experienced physician. To reduce the occurrence of posttransfusion hepatitis, it is essential to screen all blood donations for hepatitis B surface antigen by the most sensitive and specific assays. Blood donations that are found to be reactive in the HBsAg test are automatically confirmed by the HBsAg confirmatory assay. If the specimen is neutralizable in the confirmatory test, the specimen is considered positive for HBsAg. Hepatitis B surface antigen testing of donated blood has begun in 1975 (Table 1).

Currently, all blood donors are screened for HBsAg, but occasional transmission of HBV still occurs due to the inclusion of window period donations (i.e., blood from recently infected donors who are antibody negative but still viremic). Detection of early HBV infection of blood donors is still a major problem of blood transfusion. The current third-generation licensed HBsAg tests (mostly radioimmunoassay and enzyme immunoassays) cannot detect HBV in the window period for HBV infection. This is a strong motivation for introducing molecular detection techniques to the field [23]. There are some commercially available test methods for detecting HBV DNA in the market now, such as Chiron’s Quantiplex HBV DNA [24], Digene’s Hybrid Capture, Abbott’s HBV DNA assay, and Roche’s Amplicor HBV Monitor. Using these commercial hybridization or PCR-based assays, HBV DNA can be detected 1–3 weeks before the appearance of HBsAg [25]. Some chronically infected patients who have lost their HBsAg remain HBV DNA positive but are disqualified as potential blood donors. Molecular detection of HBV DNA is more sensitive than current methods employed for HBsAg screening [22, 23, 24, 25].

Antibodies to the Hepatitis B Core Antigen (Anti-HBc)

Determination of anti-HBc (total) is also used to monitor the progress of the hepatitis B viral infection. Determination of anti-HBc (IgM) is employed to distinguish an acute hepatitis B infection from a chronic infection. The anti-HBc test developed in 1987 detects an antibody to the hepatitis B virus that is produced during and after infection. If an individual has a positive anti-HBc test, but the HBsAg test is negative, it may mean that the person once had hepatitis B but has recovered from the infection. Of the individuals with a positive test for anti-HBc, many have not been exposed to the hepatitis B virus; thus, there is a frequent problem of false positives. Although the individual may be permanently deferred from donating blood, it is unlikely that the person’s health will be negatively affected. (Note: This antibody is not produced following vaccination against hepatitis B [26].

Hepatitis C Virus

The hepatitis C virus (HCV) is a member of the Flaviviridae family of viruses, which are associated with both human and animal diseases [27]. Hepatitis caused by HCV is the most common chronic blood-borne infection in the United States. Over 4 million Americans are believed to be infected. HCV can also be transmitted through blood transfusion. HCV causes inflammation of the liver, and up to 80% of those exposed to the virus develop a chronic infection, which can lead to liver inflammation, cirrhosis, cancer, and death. Eventually, up to 20% of people with HCV may develop cirrhosis of the liver or other severe liver diseases. As in other forms of hepatitis, individuals may be infected with the virus, but may not realize they are carriers since they do not have any symptoms. Because of the risk of serious illness, people with HCV need to be followed closely by a physician with experience evaluating this infection. Since the full-length HCV cDNA was first cloned in 1989, significant progress has been made in characterizing its molecular biology [13]. But, the natural history of HCV infection is still evolving, and current treatment options for patients are either limited or expensive [27]. There is no vaccine for HCV, and the current treatment includes a combination of alpha interferon and ribavirin as well as the combination of the nucleotide polymerase inhibitor sofosbuvir and the NS5A inhibitor velpatasvir [28]. Although the former is efficacious in only a minority of patients [29], the latter has been shown to be effective in a broad range of patients [28]. The life cycle of the HCV continues to be poorly understood due to the lack of an efficient cell culture system [30]. There is an urgent need to develop a highly sensitive detection method for studying possible extrahepatic sites for the replication of hepatitis C virus. We recently established a cell culture system for the replication of HCV by using human T and B leukemia cell lines [31]. This model should represent a valuable tool for the detailed study of the initial steps of the HCV replication cycle and for the evaluation of evolving antiviral molecules. Currently, appropriate vaccine strategies for HCV have not been developed. Early detection and prevention of HCV infection are most important for blood safety.

It is a formidable task to design primers and probes for sensitive nucleic acid level diagnostic assays throughout the open reading frame of the HCV genome because of a high mutation rate in this genomic region. However, the untranslated region of about 341 nucleotides contains highly conserved domains which allows for stable primer design for qualitative and quantitative diagnostic tests which have equivalent sensitivity against the known six various genotypes of HCV.

Antibodies to the Hepatitis C Virus (Anti-HCV)

In 1990, the first specific test for hepatitis C virus, the major cause of “non-A, non-B” hepatitis, was introduced. Now, a third-generation ELISA kit is available to detect antibodies to HCV, and screening blood for HCV antibodies is recommended. These assays are based on detection of serum antibody to various HCV antigens because these antibodies are nearly universally present in patients who are chronically infected with HCV [32]. The HCV screening tests are known to have significant limitations, and positive samples should be further tested by HCV confirmatory tests.

HCV Confirmatory Tests

Guidelines provided by the CDC recommend that HCV antibody screening test-positive samples should be confirmed with serologic or nucleic acid supplemental testing. HCV confirmatory tests include the recombinant immunoblot assay in which several recombinant peptide antigens are applied on a strip that is then probed with the patient’s serum. In this way, the response to individual antigens can be recognized, and some false-positive ELISA results can be eliminated (e.g., RIBA, Chiron HCV 3.0, and PCR assay) (e.g., Roche COBAS Amplicor HCV test, version 2.0). Laboratories can choose to perform this testing on all positive specimens or based on screening test-positive (signal to cutoff) ratios. The positive predictive values (s/co) can vary depending on the prevalence of infection in the population being screened.

HCV antibodies are not generally detectable for at least 6 weeks and may not appear for several months. Acute HCV infections are relatively rare among blood donors, but the antibody tests often fail to detect these patients in the window period between the time of infection and the time of appearance of antibody detectable by the above assays. High-sensitivity detection of HCV during the window period is a long-term technical challenge in the field. Tests for HCV RNA genome detection based on the PCR or other highly sensitive RNA detection systems have been used for the diagnosis of acute hepatitis [26]. Sensitive detection of HCV RNA based on RT-PCR or other nucleic acid amplification techniques can be readily accomplished with kits that are now available commercially. For example, in 1999 the FDA-approved Roche’s Amplicor HIV-1 Monitor ultrasensitive quantitative assay. It can measure HIV levels at as few as 50 copies/ml, and another commercial kit, the LCx HIV RNA quantitative assay from Abbott Laboratories, also has a detection limit at 50 copies/ml. Some studies even showed a sensitivity limit at one copy [33]. In fact, a qualitative assay should be much more sensitive than a quantitative assay for HIV/HCV screening. A sensitive qualitative HCV molecular detection assay will possibly interdict and virtually prevent all transfusion-associated HIV/HCV. The current sensitivity standard for clinical diagnostics is 100 copies per ml, but since there has been an improvement in technology, this would be the time to change sensitivity standard to 50 copies per ml.

Human Retroviruses

Antibodies to Human Immunodeficiency Virus: Types 1 and 2 (Anti-HIV-1, -2)

HIV-1 and/or HIV-2 virus cause acquired immunodeficiency syndrome or AIDS. The test is designed to detect antibodies directed against antigens of the HIV-1 or HIV-2 viruses. HIV-1 is much more common in the United States, whereas HIV-2 is prevalent in Western Africa. Donors are tested for both viruses because both are transmitted by infected blood, and a few cases of HIV-2 have been identified in US residents. In 1985, the first blood screening EIA test to detect HIV was licensed and quickly implemented by blood banks to protect the blood supply. In 1992, testing of donor blood for both HIV-1 and HIV-2 antibodies (anti-HIV-1 and anti-HIV-2) was implemented. In 1996, HIV p24 antigen testing of donated blood was mandated. Now, the p24 antigen testing is going to be compared with a PCR-based test for their ability to detect HIV in the window period.

Antibodies to Human T-Lymphotropic Virus: Types I and II (Anti-HTLV-I, Anti-HTLV-I-II)

HTLV retroviruses are endemic in Japan and the Caribbean but relatively uncommon in the United States [34]. They cause adult T-cell leukemia/lymphoma and a neurological disorder similar to multiple sclerosis. The infection can persist for a lifetime but rarely causes major illnesses in most people who are infected. In rare instances, the virus may, after many years of infection, cause nervous system disease or an unusual type of leukemia. HTLV-II infections are usually associated with intravenous drug usage, especially among people who share needles or syringes. Disease associations with HTLV-II have been hard to confirm, but the virus may cause subtle abnormalities of immunity that lead to frequent infections or rare cases of neurological disease.

In 1989, human T-lymphotropic virus antibody testing of donated blood was begun. Blood is now routinely screened for antibodies to HTLV-I/II. These test screens for antibodies directed against epitopes of the HTLV-I and HTLV-II viruses. Several commercial assays based on the enzyme-linked immunosorbent assay (ELISA) or particle agglutination formats are used for screening of HTLV antibodies, followed by confirmatory assays using Western blotting. In some infected individuals, the serologic response to HTLV infection is very low. These problems have been solved by the application of PCR amplification of specific sequences in the virus genome. PCR can be used to detect HTLV-I/II proviruses and is now the method of choice for detection of HTLV DNA directly from blood and many other tissues. Commercial PCR kits for HTLV are available [34].

West Nile Virus

The West Nile virus (WNV) is a single-stranded RNA virus of the Flaviviridae family and is one of the most recent emerging infectious disease threats to public health and, potentially, to the safety of our blood supply [35, 36, 37]. In 2002, WNV was identified as transfusion transmissible. It is transmitted by mosquitoes to birds and other animals through a mosquito bite. The virus can infect people, horses, many types of birds, and some other animals. WNV was shown in 2002 to be transmissible by blood [35], with an estimated mean risk of 2/10,000–5/10,000 in outbreak regions in the United States. The most common symptoms of transfusion-transmitted cases of WNV were fever and headache. Detection of WNV includes either a measurement of WNV antibodies or of WNV nucleic acid (detecting genetic material from the virus itself). There are two types of WNV antibody testing: IgM and IgG. In most individuals, IgM antibodies will be present within 8 days after the initial exposure to WNV, followed by IgG production several weeks later. But, the antibodies tested to detect WNV are not expedient for donor blood screening. Nucleic acid testing involves amplifying and measuring the West Nile virus’s genetic material to detect the presence of the virus in the blood or tissue. WNV NAT will be negative in the blood once clinical illness has occurred. In this situation, both NAT and IgM antibody testing may be needed. Nucleic acid tests to screen blood for WNV are commercially available and in current use. But, the viral yield for WNV infection is much lower than other viruses. Consequently, a more sensitive WNV NAT system for donor blood screening will be required, which could further reduce the risks of transfusion-transmitted WNV.

Syphilis

Serum samples from all blood units should be subjected to either the VDRL (venereal disease research laboratory) test or a treponemal test, such as the Treponema pallidum hemagglutination (TPHA) test before transfusion. Any unit found positive should be discarded as per standard safety procedures. This test is done to detect evidence of infection with the spirochete that causes syphilis. Blood centers began testing for this shortly after World War II, when syphilis rates in the general population were much higher. The risk of transmitting syphilis through a blood transfusion is exceedingly small (no cases have been recognized in this country for many years) because the infection is very rare in blood donors and because the spirochete is fragile and unlikely to survive blood storage conditions. Sensitivity and specificity of serologic tests vary depending on the type of test performed and the stage of the disease. If the donor has spirochetemia, their serologic tests are usually negative, and if the donors are antibody positive, their blood is not infectious. Syphilis serological tests for donors have less clinical significance. A nucleic acid test for accurately detecting syphilis is needed. It can be used to determine whether a blood donor is currently or has recently been infected with the spirochete.

Other Concerns

Hepatitis Viruses

In recent years, numerous infectious agents found worldwide have been identified as potential threats to the blood supply, and among these are several newly discovered hepatitis viruses that present unique challenges in assessing possible risks. Even if the hepatitis virus test is negative for all known A-E hepatitis agents, there are some unidentified hepatitis viruses, called non-A-E hepatitis viruses that can still be transmitted by blood transfusion. In the future, advances in NAT may allow rapid discovery of the unknown hepatitis viruses.

Hepatitis Delta Virus

Hepatitis delta virus (HDV) is a small RNA virus that can infect only individuals who have HBV; worldwide more than 15 million people are coinfected [38]. HDV is clinically important because it generally makes HBV infections more damaging to the liver. Increased understanding of the molecular virology of HDV will identify novel therapeutic targets for this most severe form of chronic viral hepatitis. PCR and real-time PCR methods are available for HDV RNA detection [39].

TT Virus

TT virus (TTV) [40], named for the patient from whom it was first isolated with non-A-E and G posttransfusion hepatitis in Japan in 1997, is a newly discovered transfusion-transmitted, single-stranded and circular DNA virus [41]. TTV is non-enveloped, and its entire sequence of ~3.9 kb has been determined. It is also often interpreted as a transfusion-transmitted virus [42]. At least 16 genotypes have been identified, and TTV is now found all over the world. TTV infection was sought by detection of TTV DNA in serum by polymerase chain reaction using primers generated from a conserved region of the TTV genome, e.g., the UTR region [42]. Donor blood and blood product can be screened for TTV DNA by using PCR or real-time PCR. The significance of positive findings is still unclear, because high-level TTV carriers in healthy populations are currently found [42, 43, 44]. Whether TTV actually causes hepatitis remains to be determined.

Cytomegalovirus

Cytomegalovirus (CMV) is a virus belonging to the herpes group that is rarely transmitted by blood transfusion. Donor blood is not routinely tested for CMV, and the prevalence of CMV antibody ranges from 50 to 80% of the population. But, blood contaminated with CMV can cause problems in neonates or immunocompromised patients. It also remains a major pathogen for solid-organ transplant recipients causing febrile syndromes, hepatitis, pneumonitis, retinitis, and colitis. Potential problems in selected patient populations can be prevented by transfusing CMV negative blood or frozen, deglycerolized red blood cells. Serologic tests for antibody to CMV are useful for determining whether a patient had CMV infection in the past, a determination of great clinical importance for organ and blood donors and in the pretransplant evaluation of prospective transplant recipients [45]. Commercial NAT kits are available for CMV [5], and these include the Amplicor PCR CMV Monitor Test and Hybrid Capture system CMV DNA test.

Trypanosoma Cruzi

Chagas disease is caused by the blood-borne parasite, Trypanosoma cruzi, which is transmitted to humans through insects. In the United States, Chagas disease is considered one of the neglected parasitic infections, a group of five parasitic diseases that have been targeted by CDC for public health action [46]. Commercial anti-T. cruzi assay kits are available for the qualitative detection of antibodies, Trypanosoma cruzi (T. cruzi), the causative agent of Chagas disease in human serum and plasma specimens by Abbott diagnostics [47].

Malaria

Sensitive screening tests for malaria are neither commercially available nor officially approved yet. The most effective way of screening donors is to take a proper history of malaria or of fever that could be due to malaria [48]. Donor selection criteria should be designed to exclude potentially infectious individuals from donating red blood cells for transfusion. Because there are no practical laboratory tests available to test donor blood, donors traveling to high-risk malaria areas are excluded from donating blood for 6 months. However, there is a need to develop suitable screening tests, especially for use in an endemic area. A number of clinical research approaches have been developed for the extraction, amplification, and detection of malaria parasite DNA from blood products [49].

Variant Creutzfeldt-Jakob Disease

Variant Creutzfeldt-Jakob disease [50] (vCJD, a rare but fatal brain infection) was first described in 1996 in the United Kingdom. vCJD is strongly linked with exposure to the bovine spongiform encephalopathy (BSE) agent. BSE is a transmissible spongiform encephalopathy (TSE) affecting cattle and was first reported in the United Kingdom in 1986. It has different clinical and pathologic characteristics from classic CJD. Each disease also has a particular genetic profile of the prion protein gene [51]. In recent years, questions have been raised concerning the potential risk of variant Creutzfeldt-Jakob disease for recipients of plasma-derived clotting factors, including the United States licensed factor VIII (pdFVIII), factor IX (pdFIX), and other plasma-derived products such as immune globulins and albumin. In the past 10 years, there have been some reported cases of probable variant Creutzfeldt-Jakob disease (vCJD) transmission by red blood cell transfusions in the United Kingdom [52]. Prion infections are associated with long and clinically silent incubations [50, 51]. The number of asymptomatic individuals with vCJD prion infection is unknown, posing risk to others through blood transfusion, blood products, organ or tissue grafts, and contaminated medical instruments. In order to decrease the risk, there is a need to establish a blood-based molecular assay for detection of vCJD prion infection [52]. Recently research papers have shown that sensitivity detection methods are available for vCJD prion [53, 54, 55]. However, commercial detection kits are not yet available.

Dengue Viruses

The dengue virus (DENV) is a member of the virus family Flaviviridae and is transmitted to people through the bite of an infected mosquito [56]. The dengue virus has been shown to have four subtypes. These subtypes are different strains of dengue virus that have 60–80% homology between each other. Dengue has emerged as a worldwide problem only since the 1950s. With more than one-third of the world’s population living in areas at risk for transmission, dengue infection is a leading cause of illness and death in the tropics and subtropics. According to CDC, as many as 100 million people are infected yearly [57]. Dengue is caused by any one of the four related viruses transmitted by mosquitoes. There are not yet any vaccines to prevent DENV infection, and the most effective protective measure is to avoid mosquito bites. There have been healthcare-related transmissions, including transmission by blood products [58]. Dengue infection has a viremic phase that lasts 4–8 days, and blood collected during this phase may be infective when transfused into susceptible hosts [58]. There are currently no tests for direct detection of dengue virus, but there are, however, commercial ELISA tests to detect antibodies of the dengue virus in blood samples from patients [59]. Recently, research papers have shown that PCR detection methods are available for any dengue virus strain [57, 60].

Babesia Species

Babesia is a protozoan parasite of the blood that causes a hemolytic disease known as babesiosis [61]. Babesiosis is a malaria-like parasitic disease [62], and there are over 100 species of Babesia identified [63]. In the United States, Babesia microti is the agent most commonly reported to cause human infection. Clinical confusion between human babesiosis and malaria is often reported in literature [62]. Babesia infection can also be acquired by blood transfusion [64, 65]. In fact, there have been many cases of transfusion-induced babesiosis documented [64, 65]. Risk of developing this clinical infection is increased for elderly, asplenic, or immunosuppressed patients. Current standards issued by the American Association of Blood Banks (AABB) require the indefinite deferral of a blood donor with a history of babesiosis [65]. There is a need to develop methods for identification Babesia microti in order to reduce the risk of transmission of babesiosis by transfusion. Diagnosis depends upon finding parasites on blood film examination which can be detected 2–4 weeks after a tick bite. Hamster inoculation and serology have also been used for diagnosis. The indirect fluorescent antibody test (IFAT) is available for B. microti and is the most useful serological test for early diagnosis [66]. Also, the PCR screen tests for babesiosis are technically available in the field [67].

Chagas’ Disease

Chagas disease is named after the Brazilian physician Carlos Chagas, who discovered the disease in 1909 [68]. Chagas disease is spread mainly by blood-sucking insects infected with Trypanosoma cruzi. Chagas disease can also be spread through blood transfusion, organ transplants, and from a mother to an unborn child. National screening of the blood supply [69] was instituted in early 2007 by FDA, and more than 1000 donors with T. cruzi infection have been identified within the past 3 years of testing. “Screening for T. cruzi is an important safety measure to help protect our blood supply and to help prevent the spread of Chagas disease,” says Karen Midthun, M.D., acting director of the FDA’s Center for Biologics Evaluation and Research. Currently, serological ELISA tests are available to diagnose chronic Chagas disease [70]. PCR test is not a tool for diagnosis of chronic Chagas disease in clinical practice yet, although some research results have showed that PCR is a very sensitive parasitological test for Chagas disease in active transmission regions [71]. More studies are needed for the development of this molecular method.

Severe Acute Respiratory Syndrome

Coronavirus is an RNA virus known to be associated with respiratory disease. Severe acute respiratory syndrome (SARS) is a newly recognized coronavirus whose genome sequence does not belong to any of the known coronavirus groups and which quickly spread all over the world from Asia in 2003. There has been no evidence that this infection is transmitted from blood donors to transfusion recipients, but the virus associated with SARS is present in the blood of people who are sick, and it is possible that the virus could be present in the blood immediately before a person gets sick, so that an individual with infection but no symptoms possibly could transmit SARS through a blood donation. To help determine whether or not an individual might be infected with SARS, a blood collection facility will ask a potential donor orally or in writing about any travel to a SARS-affected country or a history of SARS or possible exposure to SARS. Enzyme-linked immunoassays for detection of specific IgG and IgM antibodies and RT-PCR for detection of SARS coronavirus-specific RNA in the SARS patients have been developed. Rapid, sensitive, and specific identification of SARS and other novel coronaviruses by molecular methods will be very important in the future.

Ebola Virus

Ebola virus disease (EVD) is a rare and deadly disease caused by infection with one of the Ebolavirus species [72]. The recent outbreak in 2014–2015 is the largest Ebola outbreak since the Ebola virus was first discovered in 1976, first in Yambuku, Democratic Republic of Congo, and then in Nzara, South Sudan.

The virus family Filoviridae includes three genera: Cuevavirus, Marburgvirus, and Ebolavirus. There are five species that have been identified: Zaire, Bundibugyo, Sudan, Reston, and Taï Forest. The first three, Bundibugyo ebolavirus, Zaire ebolavirus, and Sudan ebolavirus, have been associated with large outbreaks in Africa. The virus responsible for causing the 2014 West African outbreak belongs to the Zaire species (WHO) [72].

Samples from patients are an extreme biohazard risk. Currently, a number of approaches have been developed and are available for diagnoses of Ebola virus disease: (1) antibody-capture enzyme-linked immunosorbent assay (ELISA), (2) antigen-capture detection tests, (3) serum neutralization test, (4) reverse transcriptase polymerase chain reaction (RT-PCR) assay, (5) electron microscopy, and (6) virus isolation by cell culture tests.

The WHO and FDA are working to help expedite the development and availability of medical products – such as treatments, vaccines, diagnostic tests, and personal protective equipment – with the potential to help bring the Ebola epidemic in West Africa under control as quickly as possible (FDA) [73].

Discovery of Unrecognized and Uncharacterized Viral Agents

Based on past history, it is not just a hypothetical risk that many people have been infected with unrecognized viruses, for example, many patients with symptoms of non-A-E, G, and TTV posttransfusion hepatitis. It is still possible that unexplained cases of posttransfusion hepatitis may be caused by a new, undiscovered pathogen. In recent years, numerous new infectious agents found worldwide have been identified through time-consuming procedures. By the time a new virus, such as HCV, HIV, and SARS, is found, many people are infected, and there could be a large number of fatalities. There is an urgent need to develop methods for rapid identification and characterization of previously unknown pathogenic viruses. The most recent technologies for detecting and identifying previously unrecognized pathogens are expression library screening, representational difference analysis (RDA), and broad-range polymerase chain reaction (BR-PCR). But they are all time-consuming approaches. The new unrecognized and uncharacterized viral agents can be rapidly identified by some of the new molecular approaches, e.g., subtraction hybridization [74] and DNA microarray.

Conclusion

Ensuring the safety and efficacy of blood and blood products is a critical regulatory challenge. The high safety level of the blood supply is the result of continued improvements in blood donor screening and testing. It will be achieved by introducing more updated nucleic acid tests to the field of blood safety [2, 3]. Nucleic acid testing is a method of testing blood that is more sensitive and specific than conventional tests that require the presence of antibodies to trigger a positive test result. Also, NAT works by detecting the low levels of viral genetic material present when an infection occurs but before the body develops an immune response to a virus. This improved sensitivity should enable us to significantly decrease the infection window period, allowing for earlier detection of the infection and diminishing the chances for transmission of the agent via transfusion. We are not only to protect the blood supply from known pathogens but also the emergence of new and unrecognized and uncharacterized infectious agents [4, 36, 37, 75]. The NAT methods are more sensitive and specific compared with non-NAT. In the future, NAT technology, such as PCR, may allow routine screening of donors for all the known and unknown pathogens of concern to blood safety.

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© Springer Nature Switzerland AG 2018

Authors and Affiliations

  1. 1.U.S. Food and Drug AdministrationJamaicaUSA

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