ESTABLISHED AND EMERGING

1
ESTABLISHED AND EMERGING BLOOD-BORNE
VIRUSES Juraj Petrik, PhD Head, Microbiology
RD, Scottish National Blood Transfusion Service
Honorary Senior Lecturer, University of
Edinburgh
Bratislava, 12 March 2008
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OUTLINE
Human pathogens Blood borne viruses
(BBV) Established and emerging BBV
characterization, epidemiology,
pathogenicity, treatment Highly pathogenic
global BBV Highly pathogenic endemic
BBV BBV pathogenic for certain patient
groups Pathogen detection, blood screening
and residual risk of transfusion Burden of BBV
infections global individual
3
Human pathogens
1, 407 ? 58 zoonotic 177 emerging or
reemerging Most zoonotic pathogens not
transmissible or minimally transmissible
between humans. 25 some person-to-person
transmission. Number Considered
emerging Bacteria 538 54 (10
) Fungi 317 22 (7 ) Protozoa 57 14 (25
) Helminths 287 10 (3 ) Viruses 208 77
(37 ) More than 20 families, but
Bunyaviridae, Flaviviridae, Togaviridae,
Reoviridae accounting for gt 50 MEJ
Woolhouse, S Gowtage-Sequeria, Emerging Infection
Diseases (Dec 2005)111842
4
Reasons for accelerated appearance of infectious
diseases
1 Urbanisation ? high density population areas
? poor social hygienic conditions 2
Increasing international travel ( 2.1 billion
airline passengers in 2006) 3 Increased
migration economy, result of conflict, forced
displacement 4 Increasing international trade ?
unintentional spread of pathogens, vectors.. 5
Changes in climate and habitat 6 Iatrogenic
spread of unknown pathogens 7 Emergence of
mutants, resistant strains to available drugs 8
Changes in intervention policies (e.g. widespread
insecticide use) 9 Vaccination coverage
(detrimental effect of conflicts, lack of
funding, unsubstantiated rumours etc.)
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Blood-borne viruses (BBV)
BBV can be transmitted via blood, blood
products, body fluids, cells, tissues,
organs. Established and emerging BBV
Established well understood pathogenesis,
transmission available diagnostic tests
routinely tested for (at least on a proportion of
donations). Emerging limited knowledge
regional, if any, routine testing. Highly
pathogenic global BBV HBV HIV HC
V HTLV I, II Highly pathogenic
endemic BBV WNV ? WNV Dengue
Chikungunya HAV, HEV BBV
pathogenic for certain patient groups CMV
B19 ? B19 LCMV HHV
6,8 Enterovirus ? BBV with non-
established disease association Anelloviruses,
HGV HBV - hepatitis B virus HIV - human
immunodeficiency virus HCV - hepatitis C virus
HTLV - human T-cell lymphotropic virus WNV -
West Nile virus HAV, HEV - Hepatitis A, E virus
CMV - Cytomegalo- virus B19 - Parvovirus B19
LCMV - Lymphocytic choriomeningitis virus HHV 6,
8 - Human herpesvirus 6, 8
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Highly pathogenic global BBV
HBV, HIV, HCV, HTLV I, II Typical blood-borne
viruses Blood - related transmission main
transmission route Often life-long
infection High morbidity and mortality High
global burden of infections worldwide Routinely
screened for in blood donations Low residual
risk of transfusion
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Hepatitis B virus (HBV) Virion and Genome
Virion ø42 nm enveloped (Dane
particle) Genome partially dS DNA (- strand
complete) 3.2 kb 5cohesive ends 4
overlapping orfs coding for Surface S Core
C Polymerase P (DNA poly, RT, RNase
H) Transactivating X Genotypes A -
F Replication 1 DNA repaired to fully
complementary , closed circle 2 4 RNAs
synthesized 3 3.5 kb RNA reverse
transcribed 4 One DNA species for DNA
amplification, another for virion
8
HBV Transmission, Prevalence, Infection
Transmission vertical - most prevalent
worldwide transfusion and other medical
procedures intravenous drug use sexual Prev
alence One of the most prevalent viruses 350 -
400 million chronic carriers worldwide Sub-Sahar
an Africa, most of Asia, Western Pacific 8 10
chronic inf. Amazon, Central, Eastern,
Southern Europe 2 7 India, Middle
East 5 Australia, N.Zealand, Northern
Western Europe, Northern America lt2
Infection 10 children 30-50 adults
symptomatic 2 - 10 chronic
infection 15 20 cirrhosis/Hepatocellular
carcinoma (HCC) 1 million deaths
worldwide (acute chronic hepatitis, HCC)
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HBV Infection (cont.) and Pathogenesis
Infection - continued HBsAg used as a general
marker of infection anti-HBc IgM marker of
acute infection anti-HBcIgG past or chronic
infection HBeAg indicates active replication
of virus and therefore infectiveness. Anti-HBe
virus no longer replicating. However, the
patient can still be positive for HBsAg which
is made by integrated HBV. HBV-DNA indicates
active replication of the virus, more accurate
than HBeAg especially in cases of escape
mutants. Used mainly for monitoring response
to therapy. Pathogenesis Noncythopatic
virus. Most liver damage through immune,
especially CTL responses, with contribution from
antigen-nonspecific inflammatory cells Occult
hepatitis B (after resolved acute hepatitis B, or
asymptomatic HBV exposure) Presence of HBV
DNA in serum cells of the immune system
and/or hepatic tissue Absence of serum
HBsAg Accumulating data due to the NAT
detecting low levels of HBV DNA
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HBV Prevention and Treatment
Vaccine Available since 1982- subunit later
recombinant (yeast) 95 effective in
preventing chronic infection development 116
countries - routine immunisation
programme Routine vaccination of 0-18 year
olds Vaccination of risk groups of all ages
Treatment six drugs used for the treatment
of persons with chronic hepatitis B
Adefovir dipivoxil interferon alfa-2b
pegylated interferon alfa-2a lamivudine
entecavir telbivudine
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Human immunodeficiency virus (HIV) Virion and
genome Lentivirus, Retroviridae
Virion 100 - 120 nm enveloped Genome 2
copies of non-covalently linked 9.4 kb
RNA Genotypes HIV-1 M (99.6 ), N, O
M A, B, C, D, F, G, K - subtypes E, I - CFR
(circulating recombinant forms) predominant
C 47.2 ) A and CRF02_AG 27
B 12.3 HIV-2 subtypes A - G
A (0.11 ) and B most prevalent Proteins
gag, pol, env regulatory vif, vpr, tat,
rev, nef, Vpx(HIV-2), Vpu (HIV-1)
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HIV Transmission and prevalence
Transmission unprotected sex contaminated
needles breast milk from an infected mother
to her baby at birth (20 -30 ) blood, blood
products - almost eliminated in developed
countries Prevalence WHO 2007
estimates 33.2 million people living with
HIV over 60 in sub-Saharan Africa 15
South East Asia 2.5 million new
infections 2.1 million deaths
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HIV Infection and pathogenesis
Infection CD4 CCR5 (almost all individuals
initially infected with CCR5-trophic virus
32 aminoacid deletion homozygots
protected) CXCR4 Mannose-specific C-type
lectin receptors such as DC-SIGN (dendritic
cells) Huge reservoir of replicating virus
(1010 infected cells in an average
patient) Pathogenesis Slow destruction of
T-cells, in particular central memory cells,
necessary for lifelong protection against
viruses 1 direct viral killing of infected
cells 2 increased rates of apoptosis in
infected cells 3 killing of infected CD4 T
cells by CD8 cytotoxic lymphocytes that
recognize infected cells
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HIV Prevention and Treatment
Preventative vaccine remains elusive 23 approved
drugs (USA, 2007) 8 NRTI (nucleoside or
nucleotide reverse transcriptase inhibitors)
Zidovudine, Didanosine, Zalcitabine, Stavudine,
lamivudine, Abacavir, Tenofovir,
Emtricitabine 3 NNRTI (non-nucleoside reverse
transcriptase inhibitors) Nevirapine,
Efavirenz, Delavirdine 10 PI (protease
inhibitors) Saquinavir, Indinavir, Rotonavir,
Nelfinavir, Amprenavir, Lopinavir Ritonavir,
Atazanavir, Fosamprenavir, Tripanavir,
Darunavir 2 E/F I (entry or fusion inhibitors)
Enfuvirtide, Maraviroc Need for new
drugs resistant mutants better
tolerability lower toxicity co-formulations
better treatment adherence
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Hepatitis C virus (HCV) Virus Hepacivirus
Flaviviridae
Virion Enveloped, 55 - 65 nm Genome 9.6 kb
sS RNA Coding for polyprotein,
subsequently cleaved structural C, E1, E2,
non-structural p7, NS2, NS3, NS4A,
NS4B, NS5A, NS5B Genotypes 1 -6 (gt 100
subtypes) Replication RNA poly error
rate 1 in 10,000 to 1 in 100,000
After www.med.uni-heidelberg.de
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HCV Transmission and prevalence
Transmission injectable drug use blood, blood
products vertical (3 - 15 risk) Prevalence
180 million people infected worldwide USA
3 - 4 million Europe 15 million Asia 90 -
95 million Africa 30 - 40 million Americas 12
-15 million It is estimated only about ½
diagnosed in developed countries lt1
Australia, Canada, Northern Europe 1 USA,
most of Europe gt2 Africa, Latin America,
Central South-Eastern Asia, (some countries
5 - 10 ) 19 (10 - 19 years old persons) - 60
(30 years old persons) - - Nile delta Egypt
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HCV Infection and pathogenesis
Infection CD81, SR-BI, LDL rec, L/DC-SIGN 60
- 70 asymptomatic 50 - 80 becomes
chronic Pathogenesis 10 -20 of
chronically-infected individuals develop
cirrhosis, 1 - 5 HCC HCV responsible for
27 cirrhosis 50 - 76 of all
liver cancers (25 HCC) 2/3 of all
liver transplants Mechanism both, the
immune-system-mediated pathobiological changes
and direct viral cytopathic effects
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HCV Prevention and Treatment
Prevention no vaccine In the absence of a
vaccine, all precautions to prevent infection
must be taken including ? Screening and testing
of blood and organ donors ? Virus inactivation
of plasma derived products ? Implementation and
maintenance of infection control practices in
health care settings, including appropriate
sterilization of medical and dental equipment ?
Promotion of behaviour change among the general
public and health care workers to reduce
overuse of injections and to use safe injection
practices ? Risk reduction counselling for
persons with high-risk drug and sexual practices.
Treatment pegylated interferon and
ribavirin success rate depends on
genotype 1 up to 50
23 50 - 80
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Highly pathogenic endemic viruses
Mostly emerging WNV (? WNV), Dengue,
Chikungunya, HAV, HEV Defined primarily as
arthropode-borne or mosquito-borne (WNV,
Dengue, Chikungunya) or food or water-borne
(HAV, HEV) But Emerging bloodborne pathogens
include new and re-emerging agents that can be
transmitted through blood, blood products, body
fluids, and biological therapeutic products,
including cells, tissues, and organs Increasing
territorial spread, following spread of suitable
vectors, probably as a consequence of the
climate change Significant morbidity and
mortality High global burden of infections
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West Nile Virus (WNV) Flavivirus, Flaviviridae
Virion 50 nm, enveloped Genome sS RNA -
11kb coding for structural C, E, M and
seven NS proteins Lineages 1 and 2
(recently circulating in Central
Europe) Infection Majority asymptomatic Minori
ty Mild (fever) Neuroinvasive - 10
deaths In infected individuals the ratio
between the three states is roughly
110301. Host range Mostly birds
Susceptible mammalian species horse, dog,
man Transmission Mosquito - Culex and 16 other
species Blood, blood products Prevalence Afric
a, Asia (India, Indonesia), Australia, Southern
Europe, Eastern Europe (co-circulation of 1
and 2) 1999 introduction to USA (lineage 1)
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WNV US EPIDEMICS
1999 2000 2001
2002 2003 2004
2005 2006 2007
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WNV Statistics of US epidemics
Year Overall clinical cases Viremic blood donors Fatalities
1999 62 nd 7
2000 21 nd 2
2001 66 nd 9
2002 4156 nd 284
2003 9861 818 264
2004 2539 224 100
2005 3000 417 119
2006 4269 361 177
2007 3576 332 115
nd not determined Figures from CDC
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Dengue virus Flavivirus Flaviviridae
Virion 50 nm, enveloped Genome sS RNA - 4
serotypes DENV 1 - 4 Transmission most common
mosquito-borne viral disease Aedes aegypti
(urban species) Aedes albopictus Prevalence 2
.5 billion people at risk of infection gt100
countries Africa, Americas, Eastern
Mediterranean, South-east Asia, Western
Pacific
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Dengue virus - continued
Infection Mosquito bite 2 - 7 day
viraemia Pathogenesis 50 - 100 million / year
? Dengue fever (DF) ? 1 dengue hemorragic
fever (DHF) and Dengue shock syndrome (DSS) ?
? 2.5 mortality (up to 20 without
treatment 1 with support
treatment Mechanism 2 theories 1)
Antibody-dependent enhancement (ADE)
2) Inherent virulence ADE Second infection
with a heterologous DENV serotype significantly
higher risk of developing DHF and DSS.
Pre-existing heterologous dengue antibody
recognizes and binds, but does not neutralize
the infecting virus and facilitates its
internalization via immunoglobulin Fc receptors
on the cell membrane of leukocytes, especially
macrophages. Prevention controlling vector
mosquitoes - insecticides water containers
etc. vaccine in development Treatment No
specific treatment supportive therapy - most
important is maintaining the circulating fluid
volume
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Chikungunya virus
Alphavirus, Togaviridae Virion 70 nm
enveloped Genome sS RNA 12
kb Transmission mosquito (Aedes aegypti,
albopictus) Prevalence until recently
tropical disease, spreading with the spread of
vector Reunion estimated 312,500 of
757,000 inhabitants infected (2005-07)
India ongoing 1. 400,000 in 2006
Europe increasing risk outbreak in Italy,
2007 (A.Albopictus since 1990
scattered foci in almost all regions)
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Chikungunya virus - continued
Infection self-limiting fever, headache,
weakness, rash, arthralgia (some up to
several months even years) 6 - 7 day
viraemia Prevention Treatment No vaccine
or preventative drug Measures Insect
repellent containing an DEET or another EPA-
registered active ingredient Long sleeves and
pants Screens on windows and doors, mosquito
net Elimination of mosquito breeding sites
(buckets, barrels, tyres etc)
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BBV pathogenic for certain patient groups
Immunocompromised 50 transfusion recipients
in UK have some individuals degree of
immunosuppression Pregnant Congenital
infection or infection at/shortly after birth -
risk of women miscarriage, developmental
defects CMV Screened for in a proportion of
donations. CMV-negative products given to at
risk patients B19 ? B19 Becoming established.
However, ongoing discussion on the
introduction of PCR screening. LCMV Several
episodes of fatal solid organ transplantations
HHV 6, 8 Enterovirus ?
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Cytomegalovirus (CMV) Cytomegalovirus,
Herpesviridae
Virion 120 -150 nm, enveloped Genome dS DNA,
230 kbp ? 200 genes Genotypes 2 - 6 major
subtypes when individual genes typed Variants C
omplex population of virus strains (virtually
infinite number if sufficient number of genes
analysed) Transmission Person-to-person by
direct contact Minority - shedding in urine,
saliva Blood, blood products Organ
transplantations Vertical Prevalence Almost
all people exposed to CMV when reaching
adulthood Seroprevalence 40 - 90
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CMV - continued
Infection Pathogenesis Incubation period 3 -12
weeks Lifetime infection Mostly
asymptomatic Minority - infectious
mononucleosis-like disease 1 of newly-born
infected 1 in 10 of these develops
significant illness involving nervous system
damage or developmental disabilities Immunoc
ompromised significant morbidity and
mortality fever, leucopenia, pneumonia,
gastrointestinal problems, impaired graft
function Late -stage HIV encephalitis,
retinitis Different strains may interact
differently with hosts immune system Co-infe
ction with different strains frequent in
immunocompromised, new data suggest also in
some 20 of immunocompetent Treatment Ganciclo
vir Cidofovir, Foscarnate - alternatives as the
resistant mutants appear
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Parvovirus B19 Erythrovirus, Parvoviridae
Virion 18 - 26 nm non-enveloped Genome sS
DNA 5.4 kb and - packaged 115 nt at the ends
? hairpin Codes for VP 1 - 3, NS1, 11kDa
protein, 7.5 kDa protein (?) Genotypes 1 -
3 Transmission Respiratory droplets Blood,
blood products Prevalence Seroprevalence childr
en under 5 2 15 year olds
50 adult population 80 over 60 year
olds gt90
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B 19 (continued)
Infection Fifth disease Seasonal
character maximum in spring in mild climate
countries 108 - 1014 /ml Until recently
generally accepted view of self resolving
infection within 6 months. New data (NAT)
suggest persistence in immunocompromised
persons. Possibility of reactivation during
other infections and drug treatments Pathogene
sis Immunocompromised individuals prolonged
anaemia, aplastic crisis in individuals with
haemolytic anaemia Vertical transmission anaem
ia, myocarditis ? hydrops fetalis Treatment Ge
nerally no treatment required In cases of
persistence, severe anaemia - intravenous IgGs
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LCMV Lymphocytic choriomeningitis
virus Arenavirus, Arenaviridae
Virion 50 - 300 nm enveloped Genome sS
RNA - 2 segments L coding for polymerase
Z (zinc binding) S coding for NP
(nucleoprotein) GPC (glycoprotein pr
ecursor ? GP1, 2 Strains Armstrong
immunostimulatory, clone 13 - immunosuppressive
WE , Pasteur, Traub, UBC Marseille, MX
Institute of Virology, Bratislava Transmission
Exposure to rodent aerosolised excreta Organ
transplantation 2 episodes 2003 and 2005
USA 1 episode 2007 Australia 10 out of 11
? deaths Blood, blood products? Rare
person-to-person?
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LCMV - continued
Infection Pathogenesis Acute infection
usually asymptomatic, self-limiting Grippe-like
fever, weakness, arthralgia, myalgia,
headache, nausea In some cases acute aseptic
meningitis, meningoencephalitis Pregnant
women may lead to abortion, congenital
malformations hydrocephalus, macro and
microcephaly, chorioenitis Acute
infection NP, GPs well expressed Persistent
infection NP expressed normally, GPs very
limited expression Prevalence Seroprevale
nce USA, Canada 4 - 5 Italy, Spain 2 - 3
Argentina 1 - 3.6 but Slovakia 37
.5 (anti - MX NP) Croatia 36
Treatment no specific treatment
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Pathogen detection
Antigen
Antibodies
(Glyco)protein HBsAg HCV core
ELISA, EIA Combi
Anti-HIV 1,2 Anti-HCV Anti-Treponema Anti-HTLV A
nti-HBc Anti-malaria
Nucleic acid TARGET AMPLIFICATION HCV RNA HIV1
RNA HBV DNA
Bacteria
35
PCR
After J. Mullins (April 01) http//ubik.microbiol.
washington.edu/Index.html
36
PCR vs. other amplification techniques
Method Target Signal Thermocycling
Sensitivity Product
amplification amplification

PCR Exponential No Yes High
Dispersed LCR No
Exponential Yes High Dispersed NASBA
Exponential No No High
Dispersed Branched DNA No
Exponential No Medium Localised RCA
No Linear (1 primer) No Medium
Localised No Exponential (2
pr) No High Dispersed
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Serological Methods
After J. Mullins (April 01) http//ubik.microbiol.
washington.edu/Index.html
38
Flat surface microarrays Microparticle based
microarrays
1 - 6 µm
Carboxylate Avidin-modified Hydrazide Maleimide
75 900 µm Solid, split pins
Particle encoding/decoding
Probes Nucleic acids, proteins, carbohydrates,
cells Surfaces Poly-L-lys, epoxy-silane, gold,
amino-silane
2-dye scale 100 combinations
100s to 100 000s of spots
In situ decoding
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PCR and microarray
PCR Microarray Standard Real
time Sensitivity High (10 geq) Low -
Medium Multiplexing Limited (30) Very limited
(5) High Confirmation Gel, microarray
NR PCR (for expression profiling) Protei
n assays No Yes not required
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• Pathogen blood screening Examples of assays
  used in SNBTS
• Detected target Detection Instrumental
  Signal Detection Throughput method
  platform readout limit (No of
  samples)
• HBsAg Sandwich immunoassay Abbott Prism
  Chemiluminiscence 1-10 pg/ml a) 200/hr
• Anti-HCV Sandwich immunoassay Abbott Prism
  Chemiluminiscence NA b) 200/hr
• Anti-HIV1,2 Sandwich immunoassay Abbott Prism
  Chemiluminiscence NA b) 200/hr
• Anti-treponema Agglutination Olympus CCD NA
  b) 240/hr
• HCV RNA Real time PCR In-house
  Fluorescence 29 geq/ml c) 30 50/6 hrs
  d)

Modified from Petrik J, Robb JS Microarrays and
Blood Diagnostics. InBioArrays From Basics to
Diagnostics (K.Appasani ed), Humana Press Inc.,
Totowa, NJ, 2007 215-230.
41
New Test Implementation and Declining Risk of
Viral Infections from Transfusion
Updated from AuBuchon, Birkmeyer, Busch. Ann
Intern Med 1997127904-9.
42
Risk (per unit) of transmission of major viruses
pre-NAT
MP-NAT
ID-NAT
HIV 1 1,300,000 1 1,900,000 1
3,000,000 HCV 1 230,000 1 1,600,000 1
2,300,000 HBV 1 180,000 1 210,000 1 410,000
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Summary of risks of transfusion
0
10
-
1
10
-
2
10
-
3
10
-
4
10
10
-
5
10
-
6
10
-
7
10
-
8
HIV
General anesthesia
HCV
HBV
Bacteria
Mistransfusion
Lung injury
GVHD
Cardiac
After S. Dzik, MD Blood Transfusion Service,
MGH, Boston
Metabolic risk in neonates
Under transfusion
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Global burden of BBV
Cumulative number of infections by HBV, HIV, HCV
1/10th of world population Mortality related
to HIV 2.1 million HBV 1 - 1.2
million HCV gt 1 million Chronic
infections Cost of HCV therapy 8,500 /
year Cost of HAART 12,000 - 24,000 /
year ( less in developing
countries) Cost of HBV therapy 3,500
(lamivudine) 7,400 - 9,000 (adefovir,
entecavir, telbivudine) 24,0
00 peginterferon Liver transplant 100,000 -
400,000 in developed countries 18,500 in
India Testing gt 1.3 Billion
45
Individual burden of host - microbes interactions
Adenovirus
Vaccinia virus
E coli
Human cell
ø 10 µm 0.8 x 2 µm 0.3 x 0.25 µm ø 0.075
- 0.09 µm
1000 pg 0.665 pg 0.095 pg
0.00029 pg 665 fg 95 fg
0.29 fg 1014 1015
gt1012/day
What is the cumulative metabolic burden of
simultaneously replicating microorganisms in the
host?
46
Levels of complexity of host - microbe
interactions
1 Pathogen intraspecies variability Quasispecies
heterogeneous, but closely related swarm of
viruses within the same host HCV example
error rate (1 in 10-4-5) and high turnover rate
(1012 /day) ? ? every possible
mutation in every position theoretically
generated in infected host every day 2
Pathogen - pathogen interactions
Co-infections HIV-infected individuals in
Europe 9 co-infected with HBV, 30 with
HCV generally accelerated diseases progress,
worsened prognosis HIV TTV Increased
titres of TTV with progression towards AIDS in
serum, bone marrow, spleen etc. HIV
HGV There seem to be some slowing down effect
of HGV co- infection Complex populations of
respiratory tract infections 3 Host genomic
variability Pharmacogenomics and individualised
therapy Prediction of response to particular
drugs Individualised dosing regimes 4 Drug
interactions
47
Personalised medicine and infectious diseases
Concept of clean infection with one
well-defined pathogen very simplistic Our data
set prior to therapeutic intervention is
currently extremely limited Personalised
infectious disease therapy will require Quick
analysis of the pathogen populations
present, perhaps via coupled PCR - microarray
approach Species identification Genotyping
Characterised mutants Quasispecies number
estimate if relevant Quantification Host
immune system components analysis Host
pharmacogenomics predicting drug responses At
the same time, continuous surveillance and
monitoring of the emerging and reemerging
pathogens must take place, resulting in
preventative measures and development of
vaccines and new therapeutic approaches
48
So there is a lot of work waiting for you
good luck! Thank you for your attention