?????????? Laboratory Diagnostic on HIV infection

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??????????Laboratory Diagnostic on HIV infection

• ?? ???
• ?? ??????????
• ?? 2006/12/03

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???????? Human Immnodeficiency Virus HIV
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?????????? (HIV Structure )

• ?????? (Retroviridae)?????(reverse
  transcriptase)?RNA????DNA, ?????DNA
• ???? Lentivirinae
• ????RNA Two single strand RNA
• ???Spherical
• 100120nm
• ??????????? Acquried Immunodeficiency Syndrome
  AIDS

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?????Phylogenetic tree

• Human immundeficiency Virus (HIV)
• Feline Immunodeficiency Virus (FIV)
• Simian Immunodeficiency Virus (SIV)

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???Life Cycle
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???Life Cycle

• ???? Target Cell CD4????? CD4T cell?????
  Macrophage ?????Monocytes ????? Dendritic cell
• ??CCR5 (R5), CXCR4?? (R4)
• ??Fusion
• ???Reverse Transcription RNA cDNA
• ??Integration
• ?? Transcription
• ?? Translation
• ?? Synthesis of progeny virus
• ?? Budding

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HIV ???? Genome
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HIV ???? (HIV-1?HIV-2???)
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HIV-1 ?? Virion Structure
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????? Laboratory Test

• P24??Antigen
• ?? Antibody
• ?? HIV-RNA (????Viral Load)
• CD4/CD8?????T lymphocytes??????????????1000/mm3
• ??????
• ???? Viral Culture

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?????Serological Markers
?? Detection of Antibody

• ???? latent periord
• ??? window period

??Detection of Antigen

• ???????67?
• ???????,?????????
• ????????????, ????????

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?????? ?????? Screening test
Confirmatory test

• ????
• ??????
• ????
• ?????
• ??????
• ??????

• ????? (WB)

• ????

• ?? ?

????
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??????Antigen derived

• ??????(Whole viral lysate antigen)
• ???????(Recombinant antigen)
• ?????(Synthetic peptide antigen)

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??????-Ab Screening Test

• ??????
• Axsym HIV comboRecombinant HIV (E. coli)antigens
  HIVp24 monoclonal (mouse) antibodies
• bioMerieux HIV-AbHIV-1-AgHIV-1 gp160, HIV-1
  ANT70 peptide, HIV-2 env peptide (amino acid
  592-603) anti-HIV-1-p24HIV-1/HIV-2/HIV-1 group
  O/anti-HIV-1 mixture
• Behring Anti-HIV subtype ORecombinant HIV-1,
  HIV-2 HIV-1 (Subtype O)mixture of recombinant
  HIV (env) proteins (E.coli) The proteins
  represent gp41 (HIV-1), gp41 (HIV1 Subtype O),
  gp36 (HIV-2)
• Pepti_LAV 1-2 Synthetic peptides 1, imitates an
  immunodominant epitope of gp 41, Peptides 2,
  imitates an epitope of gp36.

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????
????
?????????
????
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????-FDA Approval??
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???? Ab Confirmatory test-????? Western Blot

• ??
• ?HIV-1??????????? Virus Lysate
• ?Polyacylamide gel ???Gel Electrophoresis,
  ????????????????
• ???????? Blot on Nitrocellose

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????? Western Blot
????????????
???????????, ???????
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???????WB interpretation
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??????WB????Criteria for interpretation of WB
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???? Seroconversion
2006/04/27 ????-?? 2006/05/10
-P24,P160 2006/06/15 -p24, p120,
p160 ????????,?????????????????,???????????? ?????
????????,??????
Strip 4 Strip 5
Strip 4
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HIV ??????? Flow Chart -LSP
Anti-HIV 1/2 HIV-1 Ag (EIA)

• 
  

??N ???Incon
??P
HIV1Ag -P24 (EIA)
???????, ?????????, ?1????1??? Two assay (P) or
(Inconclusive)
??????, ???????? 2nd sample
Anti-HIV 1 2 ???? (LIA)
Anti-HIV1 ?? P ????? Both N Anti-HIV
2 ?? P
Anti-HIV 1 (WB)
anti-HIV 2 (WB)
??N ??P ???Indet

?? N ?? P ??? Indet
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HIV???????-?????????
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???????

• ??????
• ????WB????P24
• ????? P24?????
• ?????????
• O??HIV-2??
• HIV ????
• Multiparity
• Autoimmune disease
• ??????????????????

• ?????
• ???
• ??????
• ???????, ????
• ???????
• ????

• ?????
• ????
• ????

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??????
9?1????????????? ,WBp24 9?20??????, WB-P24
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??

• ??
• Anti-HIV-1/2HIV-1Ag ??
• ???
• Anti-HIV-1/2HIV-1Ag or Anti-HIV 1/2 ??
• HIV-1 Ag ?? or ??
• Anti-HIV-1 ??? ??
• ?????????????????
• ?????????????????????????

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HIV ????????Follow up

• CD4/CD8 (????? Flow Cytometry)
• CD4??T????
• CD8??T?????T????, ??????????
• CD4?HIV????????, ???????????
• CD8??????????
• EDTA-K3??,???24????????
• T????T-lymphocyte??36??????
• ?????CD4T?????8001200/mm3
• ??200/mm3 ???????????

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CD4??? CD4?????

• gt29
• 1428
• lt14

• gt500/mm3
• 200500/mm3
• lt200/mm3

• ???????

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????? Flow Cytometry

• ????????????????????? ?????????????
• ???????????????,??????????,??????,????????????????
  ,??,??????????????????,????????????? (Cell/mm3)

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?????
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T???????
WELL
???
PGL??????????
ARC ??????
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????-?????Viral Load

• ?? Assessing
• ????Disease prognosis
• ????????????Initiating evaluation of
  antiretroviral therapy????????????????????
• ?????????????Transplacental transmission

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??????Technology of Nucleic acid

• PCR ??????
• bDNA ?? DNA??????
• NASBA ????????

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????? Viral Load

• ?????? PCR
• ?????,??????,?????????????(HIV-1 RNA Amplication
  in vitro)
• ????? M A-G??
• ??Primer HIV gag??SK145/SKCC1B
• 6???????,?? -70 ºC, ??RNA??
• ?????,????????HIV-1?????
• Not screening test
• RNA??????????????????RNA, ?????????????,???50100?
  ??/mL
• ?????????????????

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?????? PCR
??????
????????
????
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RNA ??????Patients Developing AIDS or Dying of
AIDS with years, based on Initial HIV RNA Levels
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CD4 T???? ? ????
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CD4 HIV-RNA ?????
InDate CD3 CD4 CD8 HIV1Ag HIV1RQ
2006/5/ 0 0 0 N 0
2005/6/ 0 0 0 N 0
2005/10/ 1086 372 629 14700
2006/02/ 1227 400 719 10300
2006/09/ 906 332 537 18700
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HIV ???????????

• ?????
• ???????????CD4?????????
• ?????
• ???HIV??? (?????)
• ????? (ARC)
• ???? (AIDS) CD4lt200/mm3
• ????? CD4lt50/mm3

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Profile of HIV Infection
Late StageCore Ab Declines and becomes
Neg Envelope AbPositive HIV Ag Becomes positive
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HIV/AIDS??diagnosis

• ????A3, B3?C1C3???????

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????? Mutation

• HIV ???????????????????, ???????variants
  ???Strains

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?????? Purpose for typing

• ??????????????????, ????????
• ??????????
• ??HIV?????

??GAG, POL?ENV???????, ??????
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HIV Subtype

• HIV-1?HIV-2
• HIV-1
• M group (10???Ak)
• N group
• O group
• HIV-2 (??????????)
• 6??? (AF)
• HIV-2????SIV???????,???????????????????????30,???
  HIV-1????????,???40????

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HIV-1???? Distribution of Subtype

• ??(??B?, ???A?D?C?F?G)
• ???????????(B??)
• ??(A?D?)
• ??(AH?)
• ??(C??)
• ??(B?E??)
• ??(A?B?C??)
• ??(A?B??)
• ??? (B? F??)
• ?? (B?C ?E??)

????????CRF01_AE ? CRF12_BF ? B ? CRF08_BC ?B
?A1 ?A ?CRF14_BG

• ?? (CRF01_AE)
• ??????BF????

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?????????????

• ?????????????, ?????????
• ??????CRF (?CRF01_AE), ?????????????? ?cpx??
• ????CRF01_AE, CRF02_AG, CRF04_cpx??

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HIV???????
DNA?????
??PCR
PCR ????
PCR???????
????
?????????
??
?????????????
?????HIV????
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HIV????? Drug Resistance
??HIV?????????????

• ???????????
• ???????HIV????????
• ????????????

??????10?HIV-1??????????1??????? (Medscape
2006/07/21)
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??????? ???????

• ?????????
• ???????
• ?????????
• ???????
• ????????
• ?????????

• ??????
• ?????????
• ?????????
• ???????
• ????????
• ??????

????????, ??????????
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???-Inhibitor
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???? Viral Culture

• ??????????????
• ?????????, ??P3?????
• ????????????(PBMC)???????????

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???????Evaluation of Assay Verification Issues

• ???Sensitivity
• ???Specificity
• ???Reproducibility
• ??Simplicity
• ?????Cost/benefit
• ???Stability
• ???Accuracy
• ???Precision
• ????Ease of use
• ?????Positive Predictive Value ()
• ?????Negative Predictive Value ()

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??Conclusion
????????? Problems with Etiological Diagnosis

• ????????100????????
• No diagnostic test is 100 specific and
  100sensitive
• HIV-1????GAG?POL???????????,??70?????ENV???50??
  ???????????????????????
• HIV Highly variable gag and pol share 70 amino
  acid sequence homology in some strain, env is
  50. Infection is not detected