Indeterminate Serological Tests for HIV

1
Indeterminate Serological Tests for HIV

• Peter Riley, Consultant Microbiologist, Mayday
  Hospital
• Phil Rice, Consultant Virologist, St. Georges
  Hospital

2
Indeterminate tests

• Definition
• Examples
• Causes
• Management

3
Indeterminate

• Most of published literature is from USA
• Repeated reactivity in HIV EIA test with a
  Western Blot that is not negative or positive

4

• Testing Schedule in USA

EIA
Non-reactive
Reactive
Reported as negative
Western blot
Reactive
Non-reactive
Indeterminate
Reported as positive
Reported as negative
Reported as indeterminate
Repeat sample
5

• UNAIDS and WHO Recommendations for HIV testing
• Three different strategies dependent on testing
  objective and prevalence of infection
• The 3 strategies are based on the use of one to
  three ELISA and/or simple/rapid assays

6
UNAIDS and WHO Recommendations for HIV testing
7
Strategy I

• Serum/plasma is tested with one ELISA or
  simple/rapid assay
• Serum that is reactive is considered HIV antibody
  positive
• Serum that is non-reactive is considered HIV
  antibody negative

8
Strategy II

• Serum/plasma is first tested with one ELISA or
  simple/rapid assay. Any serum found reactive on
  the first assay is retested with a second ELISA
  or rapid/simple assay based on a different
  antigen preparation and/or different test
  principle (e.g. indirect/competitive)
• Serum that is reactive in both tests is
  considered HIV antibody positive
• Serum that is non-reactive in the first test is
  considered HIV antibody negative
• If the results of the two tests are discordant
  and remain discordant the serum is considered to
  be indeterminate

9
Strategy III

• As in strategy II, serum is first tested with
  one ELISA or simple/rapd assay and any reactive
  samples are retested using a different assay.
• Serum that is non-reactive in the first assay is
  considered to be antibody negative
• Strategy III requires a third test if serum is
  found reactive on the second assay.
• The three tests are based on different antigen
  preparations or test principles.
• Serum reactive in all three assays is considered
  to be antibody positive.

10
Strategies II and III

• In the selection of HIV antibody tests for use
  in strategies II and III, the first test should
  have the highest sensitivity, whereas the second
  and third tests should have a higher specificity
  than the first.

11
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12

• Testing Schedule in UK

EIA
Non-reactive
Reactive
Reported as negative
Second test
Third test
Reactive
Reactive
Discordant results indeterminate
Reported as positive
Repeat sample
13
HIV testing at Mayday

• Abbot AxSYM HIV 1/2 gO (EIA)
• Serodia particle agglutination test (HIV-1)

14
HIV testing at Mayday

• 5000 tests per annum
• 3700 ANC
• 1200 GUM
• 100 GPs, inpatients

15
ELISA
Reactive
Non-reactive Reported as anti-HIV 1/2 Negative
Serodia
Reactive Reported as anti-HIV 1/2 Presumptive
Positive
Non-reactive
Reference Lab
Reference Lab
Positive Reported as Positive with request for
2nd serum
16

• Case Study 1
• blood 06.05.01
• AxSYM S/CO 1.47 POS
• Serodia neg
• Serum sent to CPHL

17

• blood 06.05.01
• AxSYM S/CO 1.47 POS
• Serodia neg
• CPHL
• Innotest 0.092 neg
• Murex Ag/Ab 1.191 POS
• GACPAT HIV 1 0.872 neg
• GACPAT HIV-2 0.248 neg
• GACELISA (IgG) 0.417 neg
• MACELISA (IgM) 0.893 neg
• AACELISA (IgA) 0.763 neg
• HIV p24 antigen 0.885 neg

18

• blood 06.05.01 repeat 20.06.01
• AxSYM S/CO 1.47 16.7
• Serodia neg POS
• CPHL
• Innotest 0.092
• Murex Ag/Ab 1.191
• GACPAT HIV 1 0.230
• GACPAT HIV-2 0.248
• GACELISA (IgG) 0.417
• MACELISA (IgM) 0.275
• AACELISA (IgA) 1.811
• HIV p24 antigen 0.385

19

• blood 06.05.01 repeat 20.06.01
• AxSYM S/CO 1.47 16.7
• Serodia neg POS
• CPHL
• Innotest 0.092 2.12 POS
• Murex Ag/Ab 1.191 11.67 POS
• GACPAT HIV 1 0.230 5.481 POS
• GACPAT HIV-2 0.248 0.297 neg
• GACELISA (IgG) 0.417 2.37 POS
• MACELISA (IgM) 0.275 2.67 POS
• AACELISA (IgA) 1.811 3.19 POS
• HIV p24 antigen 0.385 1.97 POS

20

• Case Study 2
• ANC booking blood 23.07.01
• AxSYM S/CO 2.37 POS
• Serodia neg
• Serum sent to CPHL

21

• ANC booking blood 23.07.01
• AxSYM S/CO 2.37 POS
• Serodia neg
• CPHL
• Innotest 0.039 neg
• Murex Ag/Ab 0.965 neg
• GACPAT HIV 1 0.214 neg
• GACPAT HIV-2 0.421 neg
• GACELISA (IgG) 0.315 neg
• MACELISA (IgM) 0.410 neg
• AACELISA (IgA) 0.751 neg
• HIV p24 antigen 0.440 neg

22
Case Study 3
Abbot AxSYM HIV 1 /2 gO S/CO 11.3
POS Serodia (HIV 1) Negative
Sent to CPHL
23

Abbot AxSYM HIV 1 /2 gO S/CO 11.3
POS Serodia (HIV 1) Negative
Innotest 9.94 POS Biotest 11.04 POS GACPAT
HIV 1 0.13 GACPAT HIV 2 22.67 POS
24

• Case Study 4
• Referred to GUM by Chest Clinic
• 17.4.02
• AxSYM S/CO 7.8 POS
• Serodia POS
• Sent to CPHL

25

• Referred to GUM by Chest Clinic
• 17.4.02
• AxSYM S/CO 7.8 POS
• Serodia POS
• CPHL
• Innotest 4.092 POS
• Murex Ag/Ab 16.698 POS
• GACPAT HIV 1 0.466
• GACPAT HIV-2 0.182
• GACELISA (IgG) 1.148 POS
• MACELISA (IgM) 0.285
• AACELISA (IgA) 0.198
• HIV p24 antigen 0.976

26

• Western blot
• 17.04.02
• gag pol env HIV-2
• p17 neg p31 3 gp41 neg gp36 neg
• P24 /- p51 1 gp120/-
• P55 neg p66 3 gp160 1
• Indeterminate

27

• Referred to GUM by Chest Clinic
• 17.4.02 01.05.02
• AxSYM S/CO 7.8 POS
• Serodia POS
• CPHL
• Innotest 4.092 3.231 POS
• Murex Ag/Ab 16.698 16.843 POS
• GACPAT HIV 1 0.466 1.814 POS
• GACPAT HIV-2 0.182 0.242
• GACELISA (IgG) 1.148
• MACELISA (IgM) 0.285
• AACELISA (IgA) 0.198
• HIV p24 antigen 0.976 1.364 POS

28

• Western blot
• 01.05.02
• gag pol env HIV-2
• p17 neg p31 3 gp41 neg gp36 neg
• P24 /- p51 1 gp120/-
• P55 neg p66 3 gp160 1
• Indeterminate
• Identified as HIV 1 group O following
  phylogenetic analysis of the protease region pol

29

• Case Study 5
• ANC booking blood 07.06.02
• AxSYM S/CO 1.47 POS
• Serodia neg
• Serum sent to CPHL

30

• ANC booking blood 07.06.02
• AxSYM S/CO 1.47 POS
• Serodia neg
• CPHL
• Innotest 0.092 neg
• Murex Ag/Ab 1.191 POS
• GACPAT HIV 1 0.230 neg
• GACPAT HIV-2 0.248 neg
• GACELISA (IgG) 0.417 neg
• MACELISA (IgM) 0.275 neg
• AACELISA (IgA) 4.811 POS
• HIV p24 antigen 0.385 neg

31

• ANC booking blood 07.06.02 repeat 11.07.02
• AxSYM S/CO 1.47 POS 1.64 POS
• Serodia neg neg
• CPHL
• Innotest 0.092 0.026
• Murex Ag/Ab 1.191 1.234 POS
• GACPAT HIV 1 0.230 0.202
• GACPAT HIV-2 0.248 0.161
• GACELISA (IgG) 0.417 0.479
• MACELISA (IgM) 0.275 0.288
• AACELISA (IgA) 4.811 6.673 POS
• HIV p24 antigen 0.385 0.371

32

• Case Study 6
• ANC booking blood 18.06.02
• AxSYM S/CO 7.98 POS
• Serodia neg
• CPHL
• Innotest 0.143 neg
• Murex Ag/Ab 13.503 POS
• GACPAT HIV 1 0.205 neg
• GACPAT HIV-2 0.197 neg
• GACELISA (IgG) 0.231 neg
• MACELISA (IgM) 0.936 neg
• AACELISA (IgA) 0.248 neg
• HIV p24 antigen 0.299 neg

33

• ANC booking blood 18.06.02 Repeat 30.07.02
• AxSYM S/CO 7.98 8.96 POS
• Serodia neg neg
• CPHL
• Innotest 0.143 0.101
• Murex Ag/Ab 13.503 13.533 POS
• GACPAT HIV 1 0.205 0.175
• GACPAT HIV-2 0.197 0.238
• GACELISA (IgG) 0.231 0.323
• MACELISA (IgM) 0.936 0.139
• AACELISA (IgA) 0.248 0.404
• HIV p24 antigen 0.299 0.521

34

• Causes of Indeterminate HIV tests
• Seroconversion
• HIV 2 infection
• HIV 1 goup O infection
• Late HIV infection
• Other unusual

35
Sero-negative HIV infection

• Only seven previously described cases of true
  sero-negative HIV infection world-wide
• Due to rapid progression of HIV infection through
  to AIDS

36
Primary HIV infection illness

• 4th August 1995 Severe malaise, myalgia, fever
• 8th August Sore throat, Cervical axillary
  LNy
• 18th August Completely recovered
• Nov 1995 PCP diagnosed CD4 count 20
• AIDS
• Was primary infection in August 1995?
• Yes

37
Timing of infection

• Jun 1993 GUM clinic HIV Ab/RNA Negative
• Jan 1995 GP HIV Ab/RNA Negative
• Aug 1995 GP HIV Ab Neg/ RNA 105/mL
• Nov 1995 GUM HIV Ab wk pos/ RNA 106mL
• So, straight from primary HIV to AIDS in lt3
  months

38
Evolution of HIV antibody

• Days after IMX AxSYM Serodia Biotest
  GACEIA WB
• admisison Well.
• 0 wk pos neg neg pos wk pos Trace x3
• 14 wk pos wk pos neg pos wk pos Trace
  x3
• 24 wk pos neg neg pos wk pos Trace x1
• 161 neg neg neg pos neg neg
• 322 neg neg neg neg neg neg
• 442 neg neg neg neg neg neg
• NB After HAART sero-converted fully

39

• Causes of Indeterminate HIV tests
• Non-specific antibody reactions
• T-cell lymphoma
• multiple sclerosis
• IVDU
• liver disease
• autoimmune disease
• pregnancy
• immunisations
• sex with prostitutes
• pregnancy
• blood transfusions
• poor specimen integrity

40

• Causes of Indeterminate HIV tests
• Non-specific antibody reactions
• Autoimmune disease
• SLE
• Sjögrens
• Immunisation
• tetanus
• influenza
• Other infections
• EBV
• yeast, E. coli

41

• Causes of Indeterminate HIV tests
• Pregnancy
• Specificity of serological tests for HIV is less
  than 100
• Low positive predictive value

42

• Causes of Indeterminate HIV tests
• Pregnancy
• Since October 1998
• 25 ANC attendees at Mayday had reactive sera in
  HIV 1/2 EIA tests (Abbot AxSYM)
• Total number of ANC attendees tested was
• 11,000

43
Management

• Communication
• Between lab and clinicians
• Clinical
• Risk factors
• Clinical evidence of HIV infection
• Laboratory
• Further tests on current sera
• Availability of old sera
• Examination of later sample

44
MacKenzie WR, Davis JP, Petersen DE, Hibbard AJ,
Becker G, Zarvan BS. Multiple false-positive
serologic tests for HIV, HTLV-1, and hepatitis C
following influenza vaccination, 1991. JAMA.
19922681015-1017.   Celum CL, Coombs RW, Jones
M, Murphy V, Fisher L, Grant C, Corey L, Inui T,
Wener MH, Holmes KK. Risk factors for repeatedly
reactive HIV-1 EIA and indeterminate western
blots. Arch Intern Med. 19941541129-1137.   Ric
h JD, Dickinson BP, Spaulding A, Lafazia L,
Flanigan TP. Interpretation of indeterminate HIV
serology results in an incarcerated population. J
Acquir Immune Defic Syndr Hum Retrovirol.
199817376-379.   World Health Organization.
1999. Operational characteristics of commercially
available assays to determine antibodies to HIV-1
and/or HIV-2 in human sera. Report 11,
WHO/BTS/99.1,UNAIDS/99.5. Joint United Nations
Program on HIV/AIDS, Geneva, Switzerland.
  Rice PS, Cybulska B, Parry JV, Rowland-Jones
S, Daniels RS. Reappearance of HIV antibody in an
infected, seronegative individual after treatment
with highly active antiretroviral therapy. AIDS.
199913729-31.   Magee LA, Murphy KE, von
Dadelszen P. False-positive results in antenatal
HIV screening. CMAJ. 19991601285.   Doran TI,
Parra E. False-positive and indeterminate human
immunodeficiency virus test results in pregnant
women. Arch Fam Med. 20009924-929.   Mylonakis
E, Paliou M, Greenbough TC, Flanigan TP, Letvin
NL, Rich JD. Report of a false-positive HIV test
result and the potential use of additional tests
in establishing HIV serostatus. Arch Intern Med.
20001602386-2388.   Mylonakis E, Paliou M,
Lally M, Flanigan TP, Rich JD. Laboratory testing
for infection with human immunodeficiency virus
Established and novel approaches. Am J Med.
2000109568-576.   Ngan CCL, Thoe S-YS, Chan
K-P, Sng JEH, Ling, A-E. Alternative strategies
for confirmation of human immunodeficiency virus
infection require judicious use. J Clin Micro.
200240314-315.