HIV Diagnostic Tests. HIV Testing Algorithm at SydPath (National Reference Laboratory)

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Gerard Weaver
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HIV Diagnstic Tests HIV Testing Algrithm at SydPath (Natinal Reference Labratry) HIV1/2 Ab/Ag Cmbi is Architect HIV- 1 Ab/Ag EIA is Genscreen Sandwich EIA 4 th Generatin HIV Ab/Ag Chemiluminescene

1 HIV Diagnstic Tests HIV Testing Algrithm at SydPath (Natinal Reference Labratry) HIV1/2 Ab/Ag Cmbi is Architect HIV- 1 Ab/Ag EIA is Genscreen Sandwich EIA 4 th Generatin HIV Ab/Ag Chemiluminescene Micr particle Immunassay (CMIA; Architect) Magnetic micrparticles are bund t p24 Ag AND antibdies t HIV- 1 gp41 AND HIV- 2 gp36ab Incubated with patient sera t allw anti- p24 r HIV1/2 specific Ab t bind Wash steps are aided by the use f magnetic beads which retain ant Ag- Ab cmplexes A secndary Anti human Ab/recmbinant p24 is labelled with an enzyme e.g. HRP Additin f substrate e.g. TMB allws fr develpment f chemiluminescent prduct Readut is Optical density Psitive result is OD>1.0 Results may range int the 100s Nte the higher the OD readut the mre likely the result will be a true- psitive OD> 50 has PPV 99% and Sens 81% OD>151 has PPV 100% and Sens 68% S.Sassn

Prs: High Sensitivity>99% Fully autmated Fast Cns: Specificity 98% Can have false psitives with interfering Ab Windw perid 2 weeks HIV Ab/Ag ELISA (e.g. Genscreen, Liasn) Sandwich ELISA detecting p24 Ag AND Antibdies t HIV- 1 (but NOT HIV- 2).

HIV Sercnversin/Primary infectin prir t the frmatin f anti p24 Ab Patient will usually have detectable p24 until the develpment f anti p24 Ab n the HIV WB Measurement f p24 is used as an adjunct test

2 Prs: High Sensitivity>99% Fully autmated Fast Cns: Specificity 98% Can have false psitives with interfering Ab Windw perid 2 weeks HIV Ab/Ag ELISA (e.g. Genscreen, Liasn) Sandwich ELISA detecting p24 Ag AND Antibdies t HIV- 1 (but NOT HIV- 2). Usually run n samples that have POSITIVE results n the Architect CMIA (x2) High Sensitivity; Lwer Specificity P24 ELISA The HIV p24 Ag frms part f the viral capsid Levels f Serum p24 are very high in HIV Sercnversin/Primary infectin prir t the frmatin f anti p24 Ab Patient will usually have detectable p24 until the develpment f anti p24 Ab n the HIV WB Measurement f p24 is used as an adjunct test in sme patients where: HIV1/2/Ag/Ab screen and EIA are POSITIVE HIV Western Blt is NEGATIVE r INDETERMINANT These cases may represent early infectin/sercnversin Detectin f p24 prvides additinal evidence f early HIV infectin The HIV p24 ELISA is a Capture ELISA Plate cated with anti p24 Ab Incubated with patient sera t allw free p24 t bind Fllwing wash incubated with antip24 secndary Ab HRP S.Sassn

Fllwing washing additin f chemiluminescence substrate e.g. TMB and measurement via ptical density Nte that fr samples that have a POSITIVE initial p24 ELISA result a neutralisatin step is perfrmed.

3 Fllwing washing additin f chemiluminescence substrate e.g. TMB and measurement via ptical density Nte that fr samples that have a POSITIVE initial p24 ELISA result a neutralisatin step is perfrmed. This is because the p24 ELISAs are subject t high rates f FALSE POSITIVES due t interfering substances and/r Ab cmplexes. In the neutralisatin assay patient sera is pre- incubated with an unlabelled anti p24 Ab prir t perfrming the ELISA. This means any p24 Ag will be bund in Ab- Ag cmplexes and therefre unable t be measured by the sandwich ELISA. A reductin in the OD f 50% is cnsidered evidence f neutralisatin and therefre cnfirms anti- p24 specificity. HIV Western Blt Detects Ab t HIV- 1 (HIV- 2 n separate blt) Prs: Very high Specificity Cns: Expensive May miss HIV sercnversin, Requires peratr interpretatin/ nn autmated readut Slwer TAT S.Sassn

Classificatin f Indeterminant HIV Western Blts Grup Descriptin Interpretatin Specificity Reprting 1 Reactivity t viral bands but nt t p18, p24 r any gp bands Nn specific reactivity Lw specificity fr

4 Classificatin f Indeterminant HIV Western Blts Grup Descriptin Interpretatin Specificity Reprting 1 Reactivity t viral bands but nt t p18, p24 r any gp bands Nn specific reactivity Lw specificity fr HIV infectin Nt reprted at SydPath 2 Reactivity t viral bands including p18 but nt t p24 r any gp prteins 3 Reactivity viral bands including t p24 but nt t gp prteins 4 Reactivity t viral prteins including gp prteins but with <3 ther viral prteins P18 plus nn- specific reactivity Cnsistent with early infectin Cnsistent with late stage infectin Lw specificity fr HIV infectin Higher specificity fr HIV infectin Higher specificity fr HIV infectin Nt reprted at SydPath Reprted at SydPath with cmment abut acute/early infectin Reprted at SydPath HIV Prviral DNA Assay Detects integrated and unintegrated HIV DNA Prs: Cns: S.Sassn 2016 Very high specificity Used as gld standard fr HIV infectin Helpful in HIV diagnsis in HIV sercnversin/early infectin when HIV Ag/Ab tests are psitive, p24 ELISA is psitive but HIV Western Blt is blank. Helpful in excluding vertical transmissin in children brn t HIV+ mthers; where HIV Western Blts may appear psitive due t transplacental crssing f Anti- HIV Ab. Quantitative readut nly Nn FDA apprved Only available in 1 lab in Australia (SydPath) 4

5 HIV Viral Lad (Cbas Taqman Amplicr) There are numerus cmmercial kits fr measurement f HIV viral lad which are based n quantitative PCR methds e.g. Rche Amplicr, Abbtt Real- time HIV- 1 test and Rche Cbas Taqman HIV- 1 test (used at SydPath) The Rche COBAS Taqman test uses FRET t enhance RT- PCR and specificity The PCR reactin relies n DNA template, primers and nucletides but als the additin f a Taqman Prbe that cnsist f a 5 flurchrme and 3 quench which quenches the flurescence when in clse prximity. The Taqman prbe binds t the target sequence f the DNA. As the Taq plymerase extends the primer during each rund f DNA amplificatin it degrades the prbe releasing the flurchrme. Therefre the amunt f detected Flurescent is directly prprtinal t the munt f template DNA. S.Sassn

6 Past exam questins A patient with agammaglbulinaemia, n replacement intravenus immunglbulin therapy, requests an HIV test. Discuss the investigatin f HIV infectin in such a patient. (2013) As many HIV diagnstic tests are based n detectin f HIV- specific Ab in the patient, treatment with IVIG may interfere with diagnstic assays and indeed result in false negative results. The initial screening tests can still be perfrmed given bth 4 th generatin CMIA and EIA are based n detectin f bth HIV1/2 Ab AND Antigen and HIV- 1 Ab/Ag, respectively. A Psitive result wuld be cnsistent with HIV infectin but OD results may be lwer than expected due t the lack f patient- generated Ab. Therefre negative results need t be interpreted with cautin. The cnfirmatry test is mre difficult as the HIV western blt is based n detectin f anti- HIV Ab. Given the patient is stated as having Agammaglbulinemia (as ppsed t hypgammaglbulinemia) there is prbably little advantage in running an HIV WB as the nly Ab detected will be frm pled dnrs Instead the HIV Prviral DNA test shuld be perfrmed as the cnfirmatry test in this patient While nt FDA apprved this test is being perfrmed at a Natinal reference labratry in Australia under a special license. HIV viral lad testing e.g. Rche COBAS Taqman test is NOT licenced as an HIV diagnstic test, hwever may be used as an adjunctive test r subsequent mnitring test if abve HIV Ag/Ab screen and prviral DNA testing is psitive. The HIV Viral lad and CD4 cunt will nt be affected by agammaglbulinemia. Outline a prtcl fr the (pst- partum) assessment f pssible transmissin f HIV- 1 infectin t the child f a HIV- 1 infected wman. (2011) The fllwing tests shuld be perfrmed at Birth, 6 weeks, 3 mnths (6 and 12 mnths ptinal) and 18 mnths HIV western blt We wuld expect WB t be cmplete/mature given transplacental crss f maternal anti- HIV Ab Overtime shuld see de- evlutin f HIV WB in infant wh is uninfected HIV prviral DNA A negative result demnstrated infant in uninfected S.Sassn

7 ASID Guidelines: *Nte: ASID Guidelines differ frm standard clinical practice at SydPath where HIV WB and Prviral DNA measurement are run in parallel. S.Sassn

8 List the assays that are useful in the evaluatin f patients with suspected HIV sercnversin illness (primary HIV infectin) and their respective limitatins. (2012, 2010) Test Prs Cns 4 th Generatin CMIA HIV1/2 Ab/Ag test Fast TAT On demand testing Very high sensitivity?99% and Specificity >98% Detects Ab t HIV1 and HIV2 Quantitative 4 th Generatin EIA fr HIV Ab/Ag Fast Cheap Easy t run Quantitative P24 ELISA Detectin f free p24 in patients with psitive Ab/Ag screen but negative/indeterminant western blt Cheap Easy t run Quantitative 2 week windw perid Requires expensive instrumentatin Des nt detect HIV week windw perid High rates f false psitives Requires extra pre- treatment with nab if psitive t determine P24 specificity Slwer TAT HIV Western Blt Very high specificity >99% Separate blt available fr HIV- 2 Prviral DNA Used as reference standard fr HIV infectin Can be used t determine vertical transmissin in infants brn t HIV+ mthers Expensive Usually batched- s lnger TAT Nt fully autmatable Requires peratr interpretatin Nn quantitative/qualitative Can have indeterminant results Nn FDA apprved Only run at Natinal Reference Labratry under special licence Slwer TAT S.Sassn

Frequently Asked Questions: IS RT-Q-PCR Testing

Frequently Asked Questions: IS RT-Q-PCR Testing Questins 1. What is chrnic myelid leukemia (CML)? 2. Hw des smene knw if they have CML? 3. Hw is smene diagnsed with CML? Frequently Asked Questins: IS RT-Q-PCR Testing Answers CML is a cancer f the bld