HIV DIAGNOSIS IN TRANSFUSION

SETTINGS

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The Origin of HIV Disease

Scientists have confirmed that HIV originated in wild
chimpanzees and likely crossed over to humans in
Cameroon, West Central Africa.
Scientists theorize that the virus likely jumped to humans
through exposure to chimpanzee body fluids such as
blood during the hunting and butchering of bush meat.
The earliest known case of HIV was found in
1959 in a man from Kinshasha, Democratic
Republic of Congo.
HIV existed in the U.S. in the mid-to late 1970s.
In 1982, health officials began using the
term Acquired Immune Deficiency
Syndrome (AIDS).
In 1983, scientists discovered the virus that
causes AIDS. It is now known as HIV.
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Human Immunodeficiency Viruses

HIV-1: Global AIDS Epidemic
HIV-2: Mainly West Africa; less pathogenic
Both are retroviruses (RNA
DNA)
Primarily infect CD4-positive T-lymphocytes

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The Origin of HIV-1

P. troglodytes naturally infected
with SIVcpz
Non-pathogenic in chimp
Entry to humans around 1930
(1915-41)

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HIV-1 subtype prevalence in the world

HIV Testing Occurs in a Variety of Settings

Subtype C is dominating the epidemic

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The Origin of HIV-2

Sooty Mangabey (Cercocebus
atys) naturally infected with
SIVsm
Non-pathogenic in mangabeys
Geographic range of sooty
mangabeys coincides with HIV2 epicentres

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There are 2 types of HIV: HIV-I and HIV-II.

HIV-I

HIV-II

HIV-I and HIV-II Comparisons and Differences:

Both are comparable genetically and in viral structure.

Both cause AIDS.
Both are transmitted in the same ways.
Both have the same opportunistic infections associated with them.
Both show a similar period of antibody development, generally
detected within 2 weeks to 3-6 months.
Blood banks in the US and Canada began testing for HIV-I in March
1985 and HIV-II in June 1992.
HIV-I and II differ in their geographic distribution:
HIV-I is pandemic throughout the world.
HIV-II is epidemic to Sub-Saharan West Africa.
HIV-II may be asymptomatic for a longer period of time.

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The distribution of HIV subtypes and

recombinants is dynamic

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Be a virus, travel the world

Sources of HIV Variation

Rapid replication
~10.3x109 virions per
day

High mutation rate

~1 substitution per
genome per round

Recombination
~7-30 crossovers
per genome per
round
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How to detect HIV in all its complexes

Peak viremia: 106-108 gEq/mL
HIV RNA (plasma)

Ramp-up viremia
DT = 21.5 hrs

HIV Antibody

HIV p24 Ag

p24 Ag EIA HIV MP-NAT HIV ID-NAT -

"blip" viremia
days

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10

2nd gen

3 gen
rd

16
20

Viral set-point:
102 -105 gEq/mL

1st gen

22
30

40

50

60

70

80

90

100

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HIV Testing Occurs in a Variety of Settings

T&C
ANC

Prevent HIV

Blood
Banks

Provide ARV

Surveillance

HIV-infected

Infections

treatment to
persons

TB Clinics
Hospitals

STI Clinics

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Provide care
to HIV-affected
persons

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WHO recommendations for HIV testing strategies according

to test objective and prevalence of HIV infection
Objective of
HIV testing

Prevalence of
HIV infection

HIV testing
strategy

Transfusion/
donation safety

All prevalences

All prevalences

II

Surveillance
Clinical signs/
> 30 %
symptoms of HIV
infection
< 30 %

> 10%

II

< 10%

III

II

Diagnosis
Asymptomatic

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Strategy I
When the objective is safeguarding the blood supply, the test
selected for this strategy should preferably be a combined HIV1/HIV-2 assay which is highly sensitive.
Units of donated blood yielding reactive or indeterminate test
results must be considered as probably infected with HIV and should
be discarded according to universal safety instructions.
Strategy I is meant for testing the donations, but must not be used
for notifying donors of a positive test result.
If a blood or tissue donor is to be notified of a positive test result,
testing strategies II or III for diagnosis must be applied.
In situations where the blood centre does not have the capacity or
facilities for further testing, the donor should be referred to their
physician or to appropriate referral health services.

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Strategy I contd
Whatever the final diagnosis, donations which were
initially reactive should not be used for transfusion or
transplants. Several studies have shown that careful
selection of donors is more efficient than HIV antigen
testing in minimizing the risk of transfusion related
infections.
To prevent further spread of HIV, it is recommended that
systems are put in place to notify donors of their HIV
status. Such systems should include referral for
counseling and confirmation of HIV status where these
facilities are not available on site.

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Donor screening for HIV

Screening of donors for HIV antibodies using first
generation tests was implemented in March 1985.
Since a reactive test for anti-HIV would be grounds for
elimination of a blood donor,
The remaining risk came almost entirely from donors so
recently infected that they had not had time to produce
detectable antibody (the window).
the first-generation assays yielded estimates of 56 days
for the infectious preseroconversion window period.
In an effort to reduce this window period second- and
third generation antibody tests were developed in the late
1990s
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Donor screening for HIV

A test for HIV p24 antigen was introduced in 1995,64
which was ultimately supplanted by HIV NAT screening in
1999.39 The residual infectious window period with
current NAT is less than 1 week, and the current risk of
receiving blood infected with HIV that is non-reactive in
Screening for HIV antibodies is highly cost-effective, with
interdiction of approximately 500 infected donations
annually. In contrast NAT detects only approximately 10
window phase units each year in the United States and
consequently has a relatively high cost-effectiveness ratio
(>$1 million quality-adjusted life-year).

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HIV diagnosis

Figure 3: The HIV replication cycle.

Weiss, 2001.

Free HIV RNA

HIV proviral DNA in infected cells

Laboratory of diagnosis of HIV infection

1.Serodiagnosis:
1.1 Anti-HIV in serum/plasma:
high sensitivity & specificity for HI
V diagnosis
1.2 p24 Ag in serum/plasma:
early diagnosis during window
period
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2 . Molecular diagnosis:
2.1 Proviral HIV DNA in infected
cells: diagnosis and confirmation of
HIV
2.2 Free HIV RNA in plasma
diagnosis and viral load
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Laboratory tests
For HIV infection
HIV RNA

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Diagnostic algorithm for Bloodbank

Strategy I
Sample
HIV Screening
Ag/Ab EIA

Anti-HIV positive

Anti-HIV negative

HIV Screening
NAT

HIV RNA pos

If NAT is
feasible

HIV RNA neg

Further testing performed in reference centers or bloodbanks

If HIV Ab
pos
Confirmation testing
Ag-module: p24 assay
Ab-module: Immunoblot or Western blot

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EIA screening
Different formats have different advantages and disadvantages; it is
therefore important to know which format a particular assay is based
on.
So-called 4th generation antibody tests combine the detection of HIV
antibodies with that of viral p24 antigen, in order to detect antigen in
the blood sample prior to the formation of antibodies, thereby
reducing the "diagnostic window"
The accuracy of a test lies in the combination of two factors: the
tests sensitivity and its specificity.
For screening tests, the emphasis has to be placed on the utmost
sensitivity; any failure to identify a positive sample correctly could
have grave consequences. This high sensitivity, however, causes a
somewhat lower specificity. This means that the test result may
occasionally be a "false-positive".

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Non-specific reactivities
Due to the possibility of non-specific reactivity inherent in any assay,
it is preferable to use the term "reactive" . rather than "positive" .
screening test result thus avoiding misunderstandings.
All reactive screening test results must be confirmed by confirmatory
testing in order to exclude the risk of reporting non-specific reactivity
as "positive". Only then should one talk of a "positive HIV test"!
Important: a reactive screening test does not mean HIV
infection! Only a positive confirmatory test allows the diagnosis
of an HIV infection, and normally only such a result should be
communicated to the patient!
In addition to the obligatory safeguarding through confirmatory
testing e.g. by Western blot or immunoblot,
The serological diagnosis of an HIV infection always requires testing
of a second, independently obtained blood sample from the patient. If
at all possible, the patient should only then be informed about the
diagnosis.

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Ag-p24 testing
Testing for p24 antigen has been of value in the following situations:
(i) detecting early HIV infection in persons who have been exposed but
are seronegative,
(ii) screening blood for the detection of HIV infection before antibody is
produced,
(iii) diagnosing infection in the newborn
(iv) monitoring anti-viral therapy.

However, in countries that can afford and institute RNA testing, the
p24 antigen test offers no advantages; However it is still valuable in
countries where RNA testing cannot be performed.
Also used in western world to confirm Ag reaction in a positive HIV
Ag/Ab Screening assay when an Immunoblot or western blot is
Negative

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EIA testing
Ab > Ag/Ab

Enzyme linked immunosorbent

assays (ELISA)
1st generation sensitivity increase
Purified HIV lysates (poor
specificity)
2nd generation
Recombinant proteins and/or
synthetic peptides (poor specificity
3rd generation or sandwich ELISAs
Labelled antigen as conjugate
4th generation or DUO assays
Detect Ag and Ab without
differentiation
5th generation or DUO assays
Detect Ag and Ab with differentiation

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Confirming an HIV result

Western blot (New-Lav Blot)
Most Western blots have the antigens already being
electrophoresed (separated by molecular weight) and blotted
onto a membrane which is then cut into strips.
The HIV-1 viral antigens are therefore separated as follows:
gp160, gp120, p66, p55, p51, gp41, p31, p24, p17, and p15.
It is important to remember that non-viral proteins derived from
the host cells in which the virus was grown will also be present
on the nitrocellulose membrane (creating indeterminant results.

Niel T. Constantine & Holly Zink, Indian J Med Res 121, April 2005, pp 519-538
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HIV testing technologies after two decades of evolution

Confirming an HIV result

Immunoblot (INNO-LIA HIV I/II Score)
The principle of LIA is that it incorporates separate HIV antigens
on nitrocellulose strips so that each reaction can be visualized
separately
The difference is that in LIAs, HIV antigens are painted on the
strips rather than being electrophoresed from viral lysates; these
antigens are usually recombinant antigens and/or synthetic
peptides.
The LIA offers several advantages over the Western blot,
because the antigens are not derived from lymphocyte-cultured
viral lysates,

Niel T. Constantine & Holly Zink, Indian J Med Res 121, April 2005, pp 519-538
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2009 Innogenetics
HIV testing technologies after two decades of evolution

Advantages of LIA compared to western

blot
The LIA offers several advantages over the Western blot. Because
the antigens are not derived from lymphocyte-cultured viral lysates,
They eliminate the background from the non-specific host cell proteins,
thereby decreasing the number of indeterminate results in noninfected persons.
Also, the synthetic and recombinant antigens can be better
standardized, which minimizes kit lot-to-lot variations.
The antigens can be applied in optimal concentrations, unlike Western
blots, where poor expression of some antigens (e.g., gp41) is always a
problem.
Differentiation of HIV-1 and HIV-2, or even groups of virus (e.g., HIV-1
group O), is possible in LIA because of the ability to include antigens
from each virus.
Finally, the LIA offers better quality control because they include
controls on the strips to ensure that the procedure was performed
correctly and to aid in the grading of reactions.
Niel T. Constantine & Holly Zink, Indian J Med Res 121, April 2005, pp 519-538
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HIV testing technologies after two decades of evolution

Nucleic acid testing

How is NAT Testing Performed in Blood Screening?
In most countries NAT testing for blood screening is performed
on pooled samples (pool size varies between 1 to 96 across
countries) by one of the following methods
PCR - Polymerase Chain Reaction
Roche Cobas Ampliscreen

TMA Transcription Medicated Amplification

Chiron Geneprobe

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NAT methods
Advantages
Direct detection of viruses
Higher sensitivity than
ELISA
Closure of window period of
detection

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Limitations

High skill sets

High Technology
Infra-structure
Sample processing step yet
to be automated
Room for error
Cost of single NAT: 10X
ELISA

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NAT Testing
Will NAT Testing replace existing Immunoassay screening tests in Blood
Screening?
NO,
Small percentage of Antibody positive donors have been tested negative by
NAT tests.
It is possible that an antibody positive and NAT Negative donation might
transmit infection to the recipient.
Therefore NAT Testing will not replace current serology tests in blood
screening 1,2
So far no country has discontinued the serology screening for HBsAg, Anti HIV
and Anti HCV after implementation of NAT screening
1http://www.bloodservices.ca/CentreApps/Internet/UW_V502_MainEngine.nsf/9749ca80b75a038585256aa20060d703/708f54b29790a54585256abe0050069d?OpenDocument
2.http://72.14.203.104/search?
q=cache:63M5sxAngMoJ:www.nzblood.co.nz/site_resources/PDF_Documents/dnr_nat.pdf+will+NAT+replace+Antibody+screening&hl=en&gl=us&ct=clnk&cd=5

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Confirmed NAT Yield Donors: Number (Rate)

Region
Country

HIV

HCV

HBV
WP

Occult

20 (1 / 0.04 M)

36 (1 / 0.02 M)

AFRICA
Rep. So Africa

43 (1 / 0.03 M)

1 (1 / 0.73 M)

2 (1 / 4.1 M)

18 (1 / 0.45 M)

N/A

19 (1 / 2.28 M)

108 (1 / 0.4 M)

797 (1/0.05 M)

France

6 (1 / 2.52 M)

7 (1 / 2.16 M)

1 (1 / 0.099 M)

1 (1 / 0.099 M)

Germany

7 (1 / 4.54 M)

23 (1 / 1.37 M)

22 (1 / 1.43 M)

21 (1 / 1.49 M)

Greece

4 (1 / 0.22 M)

12 (1 / 0.07 M)

Italy

14 (1 / 0.55 M)

27 (1 / 0.4 M)

8 (2.3/1 M)

189 (55.5/1 M)

Poland

1 (1 / 3.0 M)

74 (1 / 0.08 M)

14 (4.73/1 M)

53 (17.9/1 M)

Spain

8 (1 / 0.54 M)

15 (1 / 0.46 M)

10 (1 / 1.64 M)

39 (1 / 0.04 M)

2 (1 / 12 M)

14 (1 / 1.71 M)

N/A

Canada:
(except Quebec)

1 (1 / 6.1 M)

3 (1 / 2.43 M)

N/A

Canada:
(Quebec only)

0 (0 / 1.75 M)

0 (0 / 2.25 M)

N/A

USA

51 (1 / 1.29 M)

302 (1 / 0.22 M)

N/A

ASIA - PACIFIC
Australia
Japan
EUROPE

UK: (England, Wales, No

Ireland) + Eire

89 (1 / 0.01 M)

NORTH AMERICA

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HIV infected donors in South Africa

2007-2008
1734 (0.12%) HIV infections
in 1,461,211 donations
HIV RNA +
anti-HIV
47 (2.6%)*

HIV RNA
anti-HIV +
14 (0.8%)

INNOTEST
p24 Ag
negative:
17 (0.98%)

1673 (96.5%)
HIV RNA+
anti-HIV+

INNOTEST
p24 Ag
positive:
27 (1.6%)

HIV-RNA
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anti-HIV

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Nevertheless NAT testing seen as the ultimate is

Not 100%

Elite Controllers

HIV seropositive
No detectable HIV RNA
(< 50 copies/mL) for > 2
years
Antiretroviral untreated
The proportion of
individuals who become
elite controllers is not
known, but is estimated
to be ~ 1-5%.
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Reports of HIV NAT negative

transfusion transmission
1. F. Najiouallah et al. J Med Virol 2004; 73:347-349
2. R. Phelps et al. Transfusion 2004; 44:929-933
3. S. Stramer et al. Transfusion 2003; 43 :Supplement :40-41A (abstract)
4. E.L. Delwart et al. Vox Sang 2004; 86 :171-177
5. A.E. Ling et al. JAMA 2000; 28: 4:210-214
6. M.C. Ferriera et al. Transfusion 2006; 46:156-157
7. A.R. Zanetti et al. Transfusion 2007; 47:1328-1329
8. L. Harritshoij et al. Transfusion 2008; 48 :2026-28
9. M. Schmidt et al. Vox Sang 2008; 95(Suppl 1) 56 (abstract)
10. U. Kalus et al. Transfusion 2009; 49: 435-439

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Conclusion
HIV diagnosis can be organized in many ways to reach an excellent
degree of safety within the countries possibilities
Only indirect testing
Or combination

All assays be indirect or direct test have their limitations.

Donor counseling is important to have appropriate treatment followup and prevent new infections
Donor counselling is based upon a positive confirmed result
Acutely infected donors should be referred to research programmes
focused on understanding this important phase of infection.
Thanks to Dr. Fransen K (WHO HIV reference center) and
Dr. Busch M (American Blood centers) for some slides

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