measurement of igg avidity in virus diagnosis
TRANSCRIPT
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www.helsinki.fi/yliopisto
Klaus Hedman, MD, Professor of clinical virology
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MEASUREMENT OF IgG AVIDITY IN VIRUS DIAGNOSIS
”It is the customary fate of new truths, to begin as heresies, and to end as superstitions” Thomas Huxley
Annual numbers of publications on: (IgG OR antibody) AND (avidity OR affinity) AND diagnos*
Web of Science, 2014
Days Weeks Months Years
Secondary infection
Onset of symptoms
IgG
Primary infection
Virus in blood
SERODIAGNOSIS OF VIRUS INFECTION
IgM
Persistence
IgG avidity (quality)
Ab + Ag Ab Ag Ka
Kd
Antibody (Ab) and antigen (Ag) form an immune complex
[Ab Ag] Ka
Kd [Ab] x [Ag] K = =
= AFFINITY ! AVIDITY
IgG-affinity (avidity): definition & calculation
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Acute-phase serum
Past-immunity serum
Rubella-IgG-EIA: effect of 8M urea on early vs. late IgG
Hedman and Seppälä, JCI 1988
Comparison
!! Comparison of urea, GuHCl, diethylamine (DEA) and ammonium thiocyanate: !! Urea, GuHCl and DEA distinguished the acute-phase IgGs from the past- infection IgGs similarly (though, at very different concentrations) !! …so did ammonium thiocyanate – N.B., at adjusted (>7) pH !! The effect of DEA was entirely due to its highly alkaline pH (10mm, pH 11.4)
Hedman and Seppälä, JCI 1988
Avidity indices from single (fixed) dilutions of serum
The avidity index obtained from a single dilution of serum is affected by the IgG concentration
Hedman and Seppälä, JCI 1988
Past-infection IgG (pooled)
Acute-phase IgG (pooled)
IgG end-point titration (± urea)
O.D.
Hedman et al, JID 1989
urea PBS
Avidity = titre
urea +
urea – x 100
– unaffected by IgG concentration
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The classical, robust procedure: avidity calculation via IgG titration curves drawn manually on paper
Serum #1 Serum #2
Urea +
Urea –
Urea –
Urea +
Two sera from a patient with CMV reactivation, with titre rise of high-avidity CMV-IgG
Avidity (%)
~
~
It’s the IgG end-point (urea+ vs. urea –) distance that tells the avidity (conveniently, from a table)
IgG-avidity calculation by curve-fitting mathematics: CMV
Korhonen et al, CDLI 1999
Computer software draws IgG titration curves (urea+ vs. urea –) of natural shapes
Here: 4 + 4 (urea+; urea –) dilutions of serum
Acute-phase serum
Past-infection serum
In each of these: 2 + 2 (urea+; urea –) dilutions
The curve-fitting IgG-avidity software draws IgG-titration curves of natural shapes; N.B., they can be highly variable.
EBV-VCA-IgG-avidity-EIA (in-house); our unpublished data
Korhonen et al, CDLI 1999
Comparison of avidities determined by curve-fitting math vs. end-point titration
End-point titration (reference method; 4+4 dilutions)
Cur
ve-f
ittin
g m
athe
mat
ics (
2+2
dilu
tions
)
0.0
5.0
10.0
15.0
20.0
25.0
30.0
35.0
40.0
45.0
50.0
55.0
60.0
65.0
-30 0 30 60 90 120 150 180 210 240 270 300 330 360
Time [days] after onset of symptoms
B19
IgG
avi
dity
(%)
C: AVI
Enders et al, J Clin Virol 2006;35:400-406
Maturation kinetics of B19-virus IgG avidity
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Twenty immunocompetent patients with acquired toxoplasmosis (symptomatic primary infection). IgG end-point titration (± 6M urea). Material: Dr. Roberto Rescaldani, Monza
TOXOPLASMA-IgG AVIDITY MATURATION AFTER PRIMARY INFECTION
Avidity (%)
cut-off (lower)
Months after onset of symptoms
Hedman et al, unpublished
DIAGNOSTIC CRITERIA IN IgG-AVIDITY MEASUREMENT
Low avidity: Rapid diagnosis by a single serum sample
Rise of avidity: Late diagnosis via delayed follow-up sampling
0,0
10,0
20,0
30,0
40,0
50,0
60,0
70,0
0 5 10 15 20 25 30
Age [months]!!
Patient 1Patient 2Patient 3Patient 4Patient 5Patient 6Patient 7Patient 8Patient 9Patient 10Patient 11Patient 12Patient 13Patient 14Patient 15Patient 16Patient 17Patient 18Patient 19
IgG AVIDITY IN PERINATAL DIAGNOSIS: CONGENITAL TOXOPLASMOSIS
Buffolano et al, EJCMID 2004
Weeks after clinical onset
low
borderline
high
IgG avidity in hantavirus disease – immunofluorescence assay
IgG avidity in secondary immune response: rubella revaccination
Hedman et al, J Med Virol 1989
PRIMARY AND SECONDARY VACCINE FAILURES DISTINGUISHED BY IgG AVIDITY: MEASLES, MUMPS (etc)
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... Excellent diagnostic performances were reached by sequential use of highly sensitive IgM assays and methods examining IgG avidity or stage specificity. IgA or IgM assays were less suitable for confirmation of toxoplasma-IgM positivity. This study documents the strength of test combinations in assessing the stage of toxoplasma infection.
METHOD COMBINATIONS ARE ACCURATE!
METHOD SPECIFICITY (%)
A alone 90 95 99
B alone 90 98 99
A & B together 99 99.9 99.99
Two independent methods used alone or in combination:
99 99.9 99.99
SPECIFICITY (%)
95 99
99
E.g. a high-quality in-house IgG-avidity EIA
E.g. a commercial IgM EIA
-! Sensitivity 100% -! Specificity 92.7% -! Neg predictive value 100% -! Pos predictive value 90%
A toxo-IgG-avidity assay that is gratifyingly sensitive; a high-avidity result then rules out recent primary infection.
Iatrogenic CMV-pseudodiagnosis revealed by avidity measurement
Days CMV- CMV- CMV- CMV- CMV-
from IgG IgM AVI IgE CF
BM-SCT titre (%) titre
-40 <200 -- -- <8
-2 400 -- 51.7 -- <8
12 800 -- 76.2 -- <8
36 1600
68 <200
A pediatric patient with aplastic anemia => BM-SCT
Many platelet infusions administered before and after TX
Our unpublished data
... Among EBV-seropositive patients with CMV primary infection, a large proportion (13/56, 23%) showed antibody profiles of EBV reactivation: seroconversion of VCA IgM and/or "fourfold rise of VCA IgG, together with high or intermediate avidity of VCA IgG.
Lazzarotto et al, 2008
CMV-IgG avidity assays from various sources (commercial; in-house) differ in diagnostic performance.
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H
H
H
H
L L
In competition-EIA, antigen in solution preferentially blocks high-avidity IgG (“H”)
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Comparison of Toxoplasma-avidity assays:
Architect vs. Vidas
141 sera; 87 of high and 50 of low
avidity
Our unpublished data
Comparison of Toxoplasma-avidity assays:
Labsystems vs. Architect
Our unpublished data
Labsystems
Comparison of Toxoplasma-avidity assays:
Labsystems vs. Vidas
Our unpublished data
Labsystems
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“How to set up protein-denaturing IgG-avidity assay(s)?”
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unusual solid-phase
“ABAI” = Antibody Bridge Avidity Index
An unusual avidity assay, reportedly unaffected by the IgG concentration
solid phase
antigen (immobilized)
antigen (in solution)
antigen (immobilized)
LUMINEX-BASED SUSPENSION MICROSPHERE IMMUNOASSAY (SMIA)
Advantages
Save Money
Save Time
Save Human
Resource
Multiplex testing
Microspheres (beads)
Green Laser: 532nm
Red laser: 635nm
…also qualifies for an avidity assay platform (our unpublished data)
www.helsinki.fi/yliopisto www.helsinki.fi/yliopisto Hedman & Söderlund-Venermo Research Group (2014)
Maria Lea
Visa Reza
Tingting
Elina Kalle
Klaus Yilin
Xuemeng
Benedict Mari Mafe
Arun Inga Satu
Home page: www.hi.helsinki.fi/labhedmansoderlund/
KLAUS HEDMAN et al. — PUBLICATIONS ON IgG-AVIDITY
1. Maple PAC, Hedman L, Dhanillal P, Kantola K, Nurmi V, Söderlund-Venermo M, Brown KE, Hedman K. Identification of past
from recent B19V infection in immunocompetent individuals by quantitative polymerase chain reaction (qPCR) and enzyme immunoassays (EIAs) – a dual laboratory study. J Clin Microbiol 2014:52:947-56.
2. Chen T, Tanner L, Simell V, Hedman L, Mäkinen M, Sadeghi M, Veijola R, Hyöty H, Ilonen J, Knip M, Toppari J, Simell O, Söderlund-Venermo M, Hedman K. Diagnostic methods for and clinical pictures of polyomavirus primary infections in children, Finland. Emerg Infect Dis 2014:20;689-92.
3. Sadeghi M, Aaltonen L-M, Hedman L, Chen T, Söderlund-Venermo M, Hedman K. Detection of TS polyomavirus DNA in
tonsillar tissues of children and adults: Evidence for site of viral latency. J Clin Virol 2014;59:55-8.
4. Meriluoto M, Hedman L, Tanner L, Simell V, Mäkinen M, Simell S, Mykkänen J, Korpelainen J, Ruuskanen O, Ilonen J, Knip M, Simell O, Hedman K, Söderlund-Venermo M. Association of human bocavirus 1 infection with respiratory disease in childhood follow-up study, Finland. Emerg Infect Dis 2012;18:264-71.
5. Namujju PB, Hedman L, Hedman K, Banura C, Mbidde EK, Dennison K, Byaruhanga RN, Muwanga M, Kirnbauer R, Surcel H-
M, Lehtinen M. Low avidity of human papillomavirus (HPV) type 16 antibodies is associated with increased risk of low-risk but not high-risk HPV type prevalence. BMC Research Notes 2011;4:170.
6. Lahtinen A, Kivelä P, Hedman L, Kumar A, Kantele A, Lappalainen M, Liitsola K, Ristola M, Delwart E, Sharp C, Simmonds P,
Söderlund-Venermo M, Hedman K. Serodiagnosis of primary infections with human parvovirus 4, Finland. Emerg Infect Dis 2011;17:79-82.
7. Smolander H, Koskinen JO, Vainionpää R, Meltola NJ, Lappalainen M, Hedman K, Soini AE. A novel antibody avidity
methodology for rapid point-of-care serological diagnosis. J Virol Meth 2010;166:86-91.
8. Hedman L, Söderlund-Venermo M, Jartti T, Ruuskanen O, Hedman K. Dating of human bocavirus infection with protein-denaturing IgG-avidity assays – Secondary immune activations are ubiquitous in immunocompetent adults. J Clin Virol 2010;48:43-8.
9. Enders M, Weidner A, Rosenthal T, Baisch C, Hedman L, Söderlund-Venermo M, Hedman K. Improved diagnosis of gestational
parvovirus B19 infection at the time of nonimmune fetal hydrops. J Infect Dis 2008;197:58-62.
10. Hannu T, Hedman K, Hedman L, Leirisalo-Repo M. Frequency of recent parvovirus infection in patients examined for acute reactive arthritis. A study with combinatorial parvovirus serodiagnostics. Clin Exp Rheumatol 2007;25:297-300.
11. Enders M, Schalasta G, Baisch C, Weidner A, Pukkila L, Kaikkonen L, Lankinen H, Hedman L, Söderlund-Venermo M,
Hedman K. Human parvovirus B19 infection during pregnancy - value of modern molecular and serological diagnostics. J Clin Virol 2006;35:400-6.
12. Buffolano W, Lappalainen M, Hedman L, Ciccimarra F, Del Pezzo M, Rescaldani R, Gargano N, Hedman K. Delayed
maturation of IgG avidity in congenital toxoplasmosis. Eur J Clin Microbiol Infect Dis 2004;23:825-30.
13. Kneitz RH, Schubert J, Tollmann F, Zens W, Hedman K, Weissbrich B. A new method for determination of varicella-zoster virus immunoglobulin G avidity in serum and cerebrospinal fluid. BMC Infect Dis 2004,4:33.
14. Roberts A, Hedman K, Luyasu V, Zufferey J, Bessières M-H, Blatz R-M, Candolfi E, Decoster A, Enders G, Gross U, Guy E,
Hayde M, Ho-Yen D, Johnson J, Lécolier B, Naessens A, Pelloux H, Thulliez P, Petersen E. Multicenter evaluation of strategies for serodiagnosis of primary infection with Toxoplasma gondii. Eur J Clin Microbiol Infect Dis 2001;20:467-74.
15. Paunio M, Hedman K, Davidkin I, Valle M, Heinonen OP, Leinikki P, Salmi A, Peltola H. Secondary measles vaccine failures
identified by measurement of IgG avidity: high occurrence among teenagers vaccinated at a young age. Epidemiol Infect 2000;124:263-71.
16. Korhonen MH, Brunstein J, Haario H, Katnikov A, Rescaldani R, Hedman K. A new method with general diagnostic utility for
the calculation of immunoglobulin G avidity. Clin Diagn Lab Immun 1999;6:725-8.
17. Aalto SM, Linnavuori K, Peltola H, Vuori E, Weissbrich B, Schubert J, Hedman L, Hedman K. Immunoreactivation of Epstein-Barr virus due to cytomegalovirus primary infection. J Med Virol 1998;56:186-91.
18. Herne V, Hedman K, Reedik P. Immunoglobulin G avidity in the serodiagnosis of congenital rubella syndrome.
Eur J Clin Microbiol Infect Dis 1997;16:763-6.
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19. Söderlund M, von Essen R, Haapasaari J, Kiistala U, Kiviluoto O, Hedman K. Persistence of parvovirus B19 DNA in synovial
membranes of young patients with and without chronic arthropathy. Lancet 1997;349:1063-5. 20. Söderlund M, Ruutu P, Ruutu T, Asikainen K, Franssila R, Hedman K. Primary and secondary infections by human parvovirus
B19 following bone marrow transplantation: Characterization by PCR and B-cell molecular immunology. Scand J Infect Dis 1997;29:129-35.
21. Liesenfeld O, Press C, Montoya JG, Gill R, Isaac-Renton JL, Hedman K, Remington JS. False-positive results in
immunoglobulin M (IgM) toxoplasma antibody tests and importance of confirmatory testing: the Platelia toxo IgM test. J Clin Microbiol 1997; 35:174-8.
22. Lappalainen M, Koskiniemi M, Hiilesmaa V, Ämmälä P, Teramo K, Koskela P, Lebech M, Raivio KO, Hedman K & study
group. Outcome of children after maternal primary toxoplasma infection during pregnancy with emphasis on avidity of specific IgG. Pediatr Infect Dis J 1995;14:354-61.
23. Söderlund M, Brown CS, Cohen BJ, Hedman K. Accurate serodiagnosis of B19 parvovirus infections by measurement of IgG
avidity. J Infect Dis 1995;171:710-13. 24. Polanec J, Seppälä I, Rousseau S, Hedman K. Evaluation of protein-denaturing immunoassays for avidity of immunoglobulin G
to rubella virus. J Clin Lab Anal 1994;8:16-21. 25. Hedman K, Lappalainen M, Söderlund M, Hedman L. Avidity of IgG in serodiagnosis of infectious diseases.
Rev Med Microbiol 1993;4:123-9.
26. Kallio-Kokko H, Vapalahti O, Hedman K, Brummer-Korvenkontio M, Vaheri A. Puumala virus antibody and immunoglobulin G avidity assays based on a recombinant nucleocapsid antigen. J Clin Microbiol 1993;31:677-80.
27. Lappalainen M, Koskela P, Koskiniemi M, Ämmälä P, Hiilesmaa V, Teramo K, Raivio KO, Remington JS, Hedman K.
Toxoplasmosis acquired during pregnancy: improved serodiagnosis based on avidity of IgG. J Infect Dis 1993;167:691-7. 28. Meurman O, Waris M, Hedman K. Immunoglobulin G antibody avidity in patients with respiratory syncytial virus infection.
J Clin Microbiol 1992;30:1479-84. 29. Lappalainen M, Koskela P, Hedman K, Teramo K, Ämmälä P, Hiilesmaa V, Koskiniemi M. Incidence of primary toxoplasma
infections during pregnancy in Southern Finland: a prospective cohort study. Scand J Infect Dis 1992;24:97-104. 30. Hedman K, Vaheri A, Brummer-Korvenkontio M. Rapid diagnosis of hantavirus disease with an IgG-avidity assay.
Lancet 1991;338:1353-6.
31. Hedman K, Lappalainen M, Seppälä I, Mäkelä O. Recent primary toxoplasma infection indicated by a low avidity of specific IgG. J Infect Dis 1989;159:736-40.
32. Hedman K, Hietala J, Tiilikainen A, Hartikainen-Sorri A-L, Räihä K, Suni J, Väänänen P, Pietiläinen M. Maturation of
immunoglobulin G avidity after rubella vaccination studied by an enzyme linked immunosorbent assay (avidity-ELISA) and by haemolysis typing. J Med Virol 1989;27:293-8.
33. Hedman K, Rousseau SA. Measurement of avidity of specific IgG for verification of recent primary rubella.
J Med Virol 1989;27:288-92. 34. Hedman K, Seppälä I. Recent rubella virus infection indicated by a low avidity of specific IgG. J Clin Immunol 1988;8:214-21.
35. Hedman K, Salonen E-M, Keski-Oja J, Räihä K. Single-serum radial hemolysis to detect recent rubella virus infection.
J Infect Dis 1986;154:1018-23. 36. Hedman K, Räihä K, Meurman O, Vaheri A. Altered hemolysis in single radial hemolysis from a single serum sample as an
indicator of recent primary rubella virus infection. J Med Virol 1984;13:323-30.