Infectious disease screening of blood products for prevention of transfusion-transmitted diseases

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Infectious disease screening of blood products
for prevention of transfusion-transmitted diseases

• Roni J. Bollag
• October 2005

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610.40 Test requirements. (a) Human blood and
blood components. Except as specified in
paragraphs (c) and (d) of this section, you, an
establishment that collects blood or
blood components, must test each donation of
human blood or blood component intended for use
in preparing a product, including donations
intended as a component of, or used to prepare, a
medical device, for evidence of infection due
to the following communicable disease agents (1)
Human immunodeficiency virus, type 1 (2) Human
immunodeficiency virus, type 2 (3) Hepatitis B
virus (4) Hepatitis C virus (5) Human
T-lymphotropic virus, type I and (6) Human
T-lymphotropic virus, type II.
(i) Syphilis testing. In addition to the testing
otherwise required under this section, you must
test by a serological test for syphilis under
640.5(a), 640.14, 640.23(a), 640.33(a),
640.53(a), and 640.65(b)(2) of this chapter.
PROPOSED 24th edition of Standards for Blood
Banks and Transfusion Services, for Comment
Purposes (10/7-12/7/05)
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Testing for transfusion-transmitted diseases

• HBsAg
• anti-HBc
• anti-HCV
• HCV RNA
• anti-HIV-1/2
• HIV-1 RNA
• anti-HTLV-I/II
• syphilis serology
• WNV RNA

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Historical perspective

• Pre-1985 syphilis, HBsAg
• 1985-1989 better HBsAg, HIV, HTLV,
• ALT, anti-HBc (surrogates for nonA, nonB Hep)
• 1990 added HCV, HIV-2, HTLV-II
• 1996 HIV p24 Ag testing
• 1999 HIV, HCV NAT
• 2004 WNV NAT

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Hepatitis B

• transmitted through parenteral, sexual
  exposure
• mean incubation time 90 days
• 50 of infections are symptomatic
• 1/500 infections are lethal
• 6 -10 of infections become chronic
• vaccination makes donor anti-HBs, HBsAg-,
  anti-HBc-
• risk of transmission 1/66,000

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Hepatitis C

• Transmitted through parenteral (common) or sexual
  (rare) exposure
• Most common chronic blood-borne infection in US
  with 150,000 new cases/year.
• 4 million infected in US
• Most common transfusion-transmitted disease
• Most common cause of liver transplants.
• 75 of infections become chronic
• Latency of symptomatic liver disease can be
  decades
• Antibody appears in serum 54 192 days
  post-infection
• Risk of transmission 1/103,000

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HIV

• First reports of transfusion-transmitted HIV in
  1982
• Blood product testing began in 1985
• transmitted through parenteral, sexual exposure
• Variable latency period
• Antibody titer detectable 45 days
  post-infection
• Common symptoms include night sweats, weight
  loss, diarrhea, thrush, purpura
• Infection chronic, but viral load abated with
  multi-drug therapy
• Risk of transmission 1/563,000

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Transfusion transmission risks
1100
Transmission risk, per unit
HIV
11000
HBV
110 000
HCV
1100 000
11 000 000
1998
2000
1996
1994
1992
1990
1988
1986
1984
2002
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HTLV-I/II

• Transmitted through parenteral or sexual exposure
• HTLV-I endemic in Japan and the Caribbean
• Most infections generate only mild symptoms
• HTLV-1 causes adult T-cell leukemia/lymphoma
  (0.1/year)
• neurological disorder similar to MS (0.1/year)
• Risk of transmission 1/641,000.

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West Nile Virus

• transmitted by arthropod exposure (incidental)
• birds are primary hosts
• 50nm spherical, lipid-enveloped flavivirus
• Single-stranded positive sense RNA genome (11,000
  nts)
• Encephalitis, meningites
• Asymmetric flaccid paralysis (poliomyelitis-like)
• 2002 4156 cases reported in U.S. (284
  fatalities)
• 5-10 mortality
• Initial screening by questionnaire
• Voluntary NAT testing through 2004, 1017 units
  withdrawn due to presumptive WNV infection
• Peak exposure in August-September
• Viremia 6.5 to 56.4 days

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Syphilis

• The great imitator
• transmitted by sexual exposure
• rarely transmitted by transfusion (2-3 reports in
  30 years)
• seroconversion occurs after occurs after
  spirochetemia
• untreated infection progresses through multiple
  stages
• testing is required by AABB (past 50 years)
• syphilis positivity considered surrogate marker
  for other diseases transmitted by high-risk
  behaviors
• RPR for cardiolipin or reagin (non-specific)
• Positive RPR sent for treponemal antibody testing
  (specific)

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Misperception?
Syphilis is a venereal disease that can also be
acquired by exposure to infected blood. -
introduction to eMedicine article
http//www.emedicine.com/MED/topic2224.htm
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Trends in infectious disease
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Syphilis
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RPR (non-treponemal)

• Antigen
• 0.003 cardiolipin
• 0.02 lecithin
• 0.09 cholesterol
• 10 choline chloride
• 0.01875 charcoal
• specimen not heat-inactivated
• acceptable specimens serum, plasma, cord blood
• unacceptable specimen CSF

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VDRL (non-treponemal)

• Antigen
• 0.03 cardiolipin
• 0.2 lecithin
• 0.9 cholesterol
• specimen heat-inactivated
• acceptable specimens serum, CSF, cord blood
• unacceptable specimen plasma

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FTA-ABS (treponemal)

• Antigen
• suspension of Treponema pallidum (Nichols)
  extracted from rabbit testicular tissue
• Sorbent
• cultures of nonpathogenic Reiter treponemes
• Detection
• FITC-labeled rabbit antihuman IgG

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TP-PA MHA-TP (treponemal)

• Antigen
• formalinized tanned sheep erythrocytes sensitized
  with T. pallidum (2.5 suspension)
• Sorbent
• 0.5 sheep red cell membranes
• 0.25 bovine red cell membranes
• 0.1 rabbit testicular extract
• 0.125 sonicated Reiter treponeme
• 1 normal rabbit serum
• Detection
• Agglutination

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Sensitivities and Specificities of Syphilis
serology tests
Standard Status Nontreponemal Tests
Standard Status Treponemal Tests
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Causes of false positive non-treponemal tests
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Causes of false positive treponemal tests
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AABB position on syphilis testing
STATEMENT OF THE AMERICAN ASSOCIATION OF BLOOD
BANKSBEFORE THE BLOOD PRODUCTS ADVISORY
COMMITTEE September 15, 2000 Syphilis Testing
Presented by Louis Katz, MDChair, AABB
Transfusion Transmitted Disease Committee
AABB believes the requirement for performing an
STS on each whole blood donation can safely be
eliminated based on thirty years experience
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AABB position on syphilis testingRationale

• transfusion-transmitted syphilis not recognized
  in US for 30 years
• transmissibility of T. pallidum occurs before
  the STS is reactive
• storage of blood components in refrigerators
  and improved oxygen tension likely to prevent
  transmission of spirochetes
• from 1990 to 1997, 57 of 241,800 component
  recalls were for incorrect syphilis testing

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Serological vs NAT testing

• Window period (based on serological testing)
• HCV (80d)
• HBV (56 d)
• HIV (16 d)
• Window period (based on NAT)
• HCV (10-30 d)
• HIV (10 d)

http//www.moffitt.usf.edu/pubs/ccj/v6n5/dept5.htm
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Algorithm for multiplex NAT screening
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Syphilis NAT testing

• Published PCR targets
• tpf-1
• BMP
• tmpA, tmpB
• 47-kDa protein gene
• 16S RNA
• polA
• Sensitivity
• 200 organisms/ml
• gold-standard (RIT) detects 2 organisms/ml

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Proposal for Treponema pallidum diagnostic PCR
based on the highly conserved mutL gene

• Primers
• forward CTTGAAACGCAAATGCTCAA
• reverse CCCTCGCTTCCTAACACAAG
• PCR product 197 bp
• Rationale
• bacterial mutL homologs are conserved through
  to humans and are essential for function

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Serology vs NAT testing for Treponema pallidum

• Window period for syphilis 1 3 wks
• NAT testing can identify 50 organisms (decreasing
  window to less than 1 week).
• The rabbit infectivity test (RIT), "gold
  standard" test has a detection limit of a single
  organism and can be used to confirm T. pallidum
  infection
• requires access to an animal facility
• extremely time-consuming and expensive
• Minimum TAT 90 d

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Problems with NAT testing for syphilis

• molecular biological expertise required
• special precautions to prevent cross-contamination
  
• commercially available NAT assays require more
  than 12 hours to perform
• the cost of each commercial NAT test is
  approximately 10 times that of the most expensive
  EIA test.

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Conclusion serological testing for syphilis in
blood products

• Serological testing for syphilis in individual
  donated blood products should be eliminated or
  replaced with highly specific and sensitive NAT
  testing, to be performed in multiplex algorithm
  with other NAT screens

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Bibliography

• Orton, S.L., Liu, H., Dodd, R.Y., Williams, A.E.
  (2002) Prevalence of circulating Treponema
  pallidum DNA and RNA in blood donors with
  confirmed-positive syphilis tests.Transfusion.
  42 94-99
• Palmer, H.M., Higgins, S.P., Herring, A.J.,
  Kingston, M.A. (2003) Use of PCR in the diagnosis
  of early syphilis in the United Kingdom. Sex.
  Transm. Infect. 79 479-483.
• Liu, H., Rodes, B., Chen, C.-Y., Steiner, B.
  (2001) New tests for syphilis rational design of
  a PCR method for detection of Treponema pallidum
  in clinical specimens using unique regions of the
  DNA polymerase I gene. J. Clin. Microbiol.
  391941-1946
• Harmening, D.M. (2005) Modern Blood Banking and
  Transfusion Practices, 5th ed. Philadelphia F.A.
  Davis and Co.
• Brecher, M.E., ed. (2002) AABB Technical Manual,
  14th ed. Bethesda, MD American Association of
  Blood Banks.
• http//www.aabb.org/pressroom/press_releases/prbpa
  csyph091400.htm
• http//www.moffitt.usf.edu/pubs/ccj/v6n5/dept5.htm
  
• http//www.aabb.org/About_the_AABB/Stds_and_Accred
  /stdsproposedbbts24.pdf
• http//www.emedicine.com/MED/topic2224.htm
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