dallas county naat program 1

Dallas County Health and Human Dallas County Health and Human Services LaboratoryServices Laboratory

Testing For Acute HIV InfectionTesting For Acute HIV Infection

Brian Emerson

HIV-1 RNA Testing Supervisor

Dallas County Health and Human Services Laboratory

Ph: (214)-819-1950

Email: [email protected]

Discussion TopicsDiscussion Topics

Introduction to HIV RNA NAAT: the basics Dallas County HIV Algorithm Pooling Methods Dallas County Laboratory Experience

Introduction to HIV RNA TestingIntroduction to HIV RNA Testing

Dallas County Laboratory utilizes the Aptima Dallas County Laboratory utilizes the Aptima HIV-1 RNA Qualitative Assay (manufactured by HIV-1 RNA Qualitative Assay (manufactured by Gen-Probe Inc.) for detection of acute HIV Gen-Probe Inc.) for detection of acute HIV infection (AHI).infection (AHI).

Aptima HIV-1 RNA Qualitative Assay is Aptima HIV-1 RNA Qualitative Assay is approved by the FDA for a diagnostic test and approved by the FDA for a diagnostic test and can be used in place of Western Blot.can be used in place of Western Blot.


Aptima HIV-1 RNA Assay is an Aptima HIV-1 RNA Assay is an in vitroin vitro nucleic acid-based amplification test (NAAT) nucleic acid-based amplification test (NAAT) used for the detection of HIV-1 RNA in used for the detection of HIV-1 RNA in human plasma and serumhuman plasma and serum

The presence of HIV-1 RNA in the plasma or The presence of HIV-1 RNA in the plasma or serum of a patient without antibodies to HIV-1 serum of a patient without antibodies to HIV-1 is an indication of acute HIV-1 infectionis an indication of acute HIV-1 infection

Introduction to HIV RNA TestingIntroduction to HIV RNA Testing The NAAT assay contains four main sections:The NAAT assay contains four main sections:

Target Capture – RNA of virusTarget Capture – RNA of virus TMA (Transcription-Mediated Amplification)TMA (Transcription-Mediated Amplification) HPA (Hybridization Protection Assay)HPA (Hybridization Protection Assay) DKA (Dual Kinetic Assay)DKA (Dual Kinetic Assay)

Gen-Probe Aptima HIV-1 & HCV Assay Training Manual


The RNA is isolated from the specimen via the use of The RNA is isolated from the specimen via the use of target capture, which helps reduce false negatives by target capture, which helps reduce false negatives by removing inhibitors. A detergent is used to solubilize removing inhibitors. A detergent is used to solubilize the viral envelope, denature proteins, and release the the viral envelope, denature proteins, and release the viral genomic RNA.viral genomic RNA.

Specific regions of the HIV-1 will be hybridized to Specific regions of the HIV-1 will be hybridized to the HIV-1 RNA target, if present, in the test specimenthe HIV-1 RNA target, if present, in the test specimen

The hybridized target is captured onto magnetic micro The hybridized target is captured onto magnetic micro particles that are separated from the serum in a particles that are separated from the serum in a magnetic field. All other extraneous components magnetic field. All other extraneous components from the reaction tube will be washed away during the from the reaction tube will be washed away during the wash cycle.wash cycle.


TMA is a transcription-based nucleic acid TMA is a transcription-based nucleic acid amplification method that utilizes two enzymesamplification method that utilizes two enzymes

Amplification of nucleic acid target producing Amplification of nucleic acid target producing amplified RNA product (amplicon)amplified RNA product (amplicon) RNA RNA DNA DNA RNA RNA

Combined with HPA detection in a single tube formatCombined with HPA detection in a single tube format Hybridization of DNA probe with ampliconHybridization of DNA probe with amplicon Degradation of label on unbound probeDegradation of label on unbound probe Detection of chemiluminescent label on DNA probeDetection of chemiluminescent label on DNA probe

Measured in Relative Light Unit (RLU)Measured in Relative Light Unit (RLU)

Gen-Probe Aptima HIV-1 & HCV Assay Training Manual Gen-Probe Aptima HIV-1 & HCV Assay Training Manual


Negative Specimen

Positive Specimen

Dallas County HIV AlgorithmDallas County HIV AlgorithmBlood sample received for HIV test

ELISA or EIA is performed

EIA + (Test repeated X2) EIA Negative

Western Blot

Western Blot Positive

Western Blot Indeterminate

Western Blot Negative

HIV Positive

NAAT Testing

NAAT Testing

NAAT Positive

NAAT Negative

AHI Protocol

NAAT Positive

NAAT Negative

Pooled NAAT Testing

NAAT Positive

AHI Protocol

Repeat HIV testing in 1 month


AHI Protocol

NAAT Negative

HIV – (Retest based on risk factors)

Bio-Rad Multispot HIV-1/HIV-2 Rapid Test

Dallas County HIV AlgorithmDallas County HIV AlgorithmBlood sample received for HIV

test


EIA Negative

Pooled NAAT Testing

NAAT Positive

AHI Protocol

NAAT Negative

HIV Negative (Retest based on risk factors)

Dallas County HIV AlgorithmDallas County HIV AlgorithmBlood sample received for HIV test


EIA + (Test repeated X2)

Western Blot

Western Blot Positive

Western Blot Indeterminate

HIV Positive

NAAT Testing

NAAT Positive

NAAT Negative

AHI Protocol



Dallas County HIV AlgorithmDallas County HIV AlgorithmBlood sample received for

HIV test


EIA + (Test repeated X2)

Western Blot

Western Blot Negative

NAAT Testing

NAAT Positive

NAAT Negative

AHI Protocol



Pooling MethodsPooling Methods The ability to combine specimens into pools The ability to combine specimens into pools

greatly reduces the cost for detection of greatly reduces the cost for detection of acute HIV infection. acute HIV infection.

Pooling specimens will reduce the cost by Pooling specimens will reduce the cost by 90%. (using 30,000 specimen per year for 90%. (using 30,000 specimen per year for analysis)analysis)

Pooling is not part of the package insert. The Pooling is not part of the package insert. The pooling method was verified and validated pooling method was verified and validated within the Dallas County Health Laboratory.within the Dallas County Health Laboratory.

The pooling procedure is performed The pooling procedure is performed manuallymanually

Pooling MethodsPooling Methods The specimens requesting HIV testing The specimens requesting HIV testing

are first tested using either the are first tested using either the BioRad HIV -1/2 Plus O EIA assay or BioRad HIV -1/2 Plus O EIA assay or Clearview Rapid HIV method (used in Clearview Rapid HIV method (used in the STAT lab and Mobile Vehicle).the STAT lab and Mobile Vehicle).

The specimens are separated into The specimens are separated into High Risk or Low Risk categoryHigh Risk or Low Risk category

High or Low Risk is determined by High or Low Risk is determined by the patient risk factors and submitter. the patient risk factors and submitter. (Ex. STD Clinic = High Risk, Family (Ex. STD Clinic = High Risk, Family Planning Clinic = Low Risk)Planning Clinic = Low Risk)

Pooling MethodsPooling Methods Currently utilizing pools of 10 for all high risk EIA Currently utilizing pools of 10 for all high risk EIA

and Rapid Nonreactive (NR) specimens.and Rapid Nonreactive (NR) specimens. Pooling is performed same day as the EIAPooling is performed same day as the EIA Nonreactive results are available 1 day after draw dateNonreactive results are available 1 day after draw date Reactive results are available 3 days after draw date Reactive results are available 3 days after draw date

Testing only High Risk specimens Testing only High Risk specimens due to costdue to cost

Western Blot Nonreactive and Indeterminate and Western Blot Nonreactive and Indeterminate and AHI suspects are tested individually.AHI suspects are tested individually.

Pooling MethodsPooling Methods

10 individual specimens 10 individual specimens are assigned to a are assigned to a specific pool numberspecific pool number

100 100 μμl of serum is l of serum is pipetted into labeled pipetted into labeled pool tubepool tube

Pool of 10 individual Pool of 10 individual specimenspecimen

100μμll of serum removed

Pool Tube 1.0ml of serum


Example specific pool numberspecific pool number: High-042110-1001 : High = High Risk Specimens, 042110= date, Pool # to date

If specific pool result is nonreactive then all result is nonreactive then all specimens assigned to the pool are reported as specimens assigned to the pool are reported as nonreactive. nonreactive.

A nonreactive result means no HIV-1 RNA detected A nonreactive result means no HIV-1 RNA detected in pool tubein pool tube


If pool result is reactive then the pool will be broken If pool result is reactive then the pool will be broken back down to 10 individual specimens and tested back down to 10 individual specimens and tested individuallyindividually

Once the reactive specimen is determined, one more Once the reactive specimen is determined, one more individual test will be performed before the specimen individual test will be performed before the specimen will be reported as an acute infectionwill be reported as an acute infection

The result of the specimen being reported as an acute The result of the specimen being reported as an acute infection will be given to the Medical Director (STD infection will be given to the Medical Director (STD Clinic), DIS Manager, and Aids SurveillanceClinic), DIS Manager, and Aids Surveillance

Pooling MethodsPooling Methods Limit of Detection with a pool of 10 Limit of Detection with a pool of 10

is 300 copies of HIV-1 RNA per mlis 300 copies of HIV-1 RNA per ml This means the specimen must have This means the specimen must have

300 copies per milliliter for the 300 copies per milliliter for the assay to detect the presence of HIV-assay to detect the presence of HIV-1 RNA1 RNA

Limit of Detection for testing an Limit of Detection for testing an individual sample is 30 copies per individual sample is 30 copies per millilitermilliliter

Dallas County Laboratory ExperienceDallas County Laboratory Experience


Reginald

Karen


Daniel Serinaldi Shayma Haq


Dallas County Health Laboratory Dallas County Health Laboratory experienced a 44% increase in experienced a 44% increase in patients requesting a HIV test from patients requesting a HIV test from 2007 to 20092007 to 2009

Dallas County 2010 projection will Dallas County 2010 projection will be over 60,000 specimens for HIV be over 60,000 specimens for HIV testing alonetesting alone


The HIV RNA program went live in The HIV RNA program went live in July 2009July 2009

From July 2009 to April 2010 a total From July 2009 to April 2010 a total of 30,617 High-Risk specimens of 30,617 High-Risk specimens testedtested

26 Acute infections were discovered 26 Acute infections were discovered 24 in Dallas County and surrounding 24 in Dallas County and surrounding

areaarea 2 from outside Dallas County2 from outside Dallas County


19% (5/26) of the AHI tested positive for Syphilis19% (5/26) of the AHI tested positive for Syphilis 15% (4/26) of the AHI tested positive for other 15% (4/26) of the AHI tested positive for other

STDs excluding Syphilis (ex. Gonorrhea)STDs excluding Syphilis (ex. Gonorrhea) 35% (9/26) of the AHI did not test positive for any 35% (9/26) of the AHI did not test positive for any

other STDs besides HIVother STDs besides HIV 31% (8/26) unknown if any other STDs were 31% (8/26) unknown if any other STDs were

presentpresent


YearTotal Specimen Tested for HIV

Confirmed by Western Blot

Confirmed by HIV-1 RNA ŧ

% Increase

2009 57060 581 9 1.5

2010* 21108 174 12 6.9

* Thru April 1, 2010 ŧ Western Blot Indeterminate specimens not included


Acute HIV-1 InfectionsAcute HIV-1 Infections 21 were BioRad HIV ½ Plus O EIA or 21 were BioRad HIV ½ Plus O EIA or

Clearview Rapid HIV Nonreactive Clearview Rapid HIV Nonreactive and Western Blot Nonreactive (No and Western Blot Nonreactive (No Bands Present)Bands Present)

5 were Western Blot Indeterminate5 were Western Blot Indeterminate 1 with only p241 with only p24 1 with p24 and p51/p551 with p24 and p51/p55 3 with gp160 3 with gp160


BioRad Western Blot Package insert

Western Blot Bands

Major Bands: p24, gp160, gp120, gp41

Western Blot: Reactive (2 major bands Present)

Western Blot: Indeterminate (Only one major band, and/or any minor bands )


HIV-1 RNA (NAAT): NonreactiveHIV-1 RNA (NAAT): Nonreactive EIA Assay: ReactiveEIA Assay: Reactive Nonreactive and Indeterminate Western BlotsNonreactive and Indeterminate Western Blots

Western Blot:Western Blot:63% (22/35) were Nonreactive for Western Blot (No Bands Present)63% (22/35) were Nonreactive for Western Blot (No Bands Present)

34% (12/35) were Indeterminate with only the p24 band34% (12/35) were Indeterminate with only the p24 band

3% (1/35) were Indeterminate with only the gp160 band3% (1/35) were Indeterminate with only the gp160 band

Interesting once the p24 band will be used for Interesting once the p24 band will be used for AHI detection in the 4AHI detection in the 4thth Generation EIA Generation EIA


Some Interesting Cases:Some Interesting Cases: Blood drawn on 03/17/10 and again on 03/23/10Blood drawn on 03/17/10 and again on 03/23/10

03/17/1003/17/10 03/23/1003/23/10

Clearview Rapid: Clearview Rapid: NRNR BioRad EIA: BioRad EIA: RR

Western Blot: Western Blot: NRNR Western Blot: Western Blot: IndeterminateIndeterminate

No Bands PresentNo Bands Present gp160gp160

HIV-1 RNA: HIV-1 RNA: RR HIV-1 RNA: HIV-1 RNA: RR

FIRST BAND TO APPEAR ON THE WESTERN BLOT: gp160


01/05/1001/05/10 01/15/1001/15/10

BioRad EIA: BioRad EIA: NRNR BioRad EIA:BioRad EIA: R R

Western Blot: Western Blot: NRNR Western Blot: Western Blot: RR

No Bands PresentNo Bands Present gp160, p24 Bands Presentgp160, p24 Bands Present

HIV-1 RNA: HIV-1 RNA: RR HIV-1 RNA: HIV-1 RNA: RR

Some Interesting Cases:Some Interesting Cases:Blood drawn on 01/05/10 and again on 01/15/10Blood drawn on 01/05/10 and again on 01/15/10


Blood DrawnBlood Drawn 04/08/1004/08/10

BioRad EIA BioRad EIA R (x2)R (x2)

Western BlotWestern Blot RR

Bands PresentBands Present All bands All bands presentpresent

HIV-1 RNA (Dallas)HIV-1 RNA (Dallas)

HIV-1 RNA (Houston)HIV-1 RNA (Houston)

NRNR

NRNR

Interesting Case:Interesting Case:

Possible reason: If patient is already on antiretroviral medication Possible reason: If patient is already on antiretroviral medication then the viral load will be below the detectable range.then the viral load will be below the detectable range. The patient’s history is now being investigated for a possible The patient’s history is now being investigated for a possible explanation for the HIV-1 RNA test result.explanation for the HIV-1 RNA test result.


01/21/1001/21/10 02/04/1002/04/10

Clearview Rapid: Clearview Rapid: NRNR

BioRad EIA: BioRad EIA: NRNR

Clearview Rapid: Clearview Rapid: NRNR

BioRad EIA: BioRad EIA: NRNR

Western Blot: Western Blot: NRNR Western Blot: Western Blot: NRNR

No Bands PresentNo Bands Present No Bands PresentNo Bands Present

HIV-1 RNA: HIV-1 RNA: RR HIV-1 RNA: HIV-1 RNA: NRNR

One possible false positive

Specimen also tested Positive for Syphilis on 01/21/10 and 02/04/10

Possible False Positive: 1 out of 30,617

Solution: Testing the tube for Syphilis (which is not in stored in the NAAT testing area) to make sure the HIV tube was not contaminated.


Dallas County Health and Human Dallas County Health and Human Services Laboratory tests Nonreactive Services Laboratory tests Nonreactive and/or indeterminate Western Blots for and/or indeterminate Western Blots for other Public Health Departments other Public Health Departments throughout Texas.throughout Texas.

If anyone is interested in sending Dallas If anyone is interested in sending Dallas County Laboratory specimens for HIV-1 County Laboratory specimens for HIV-1 RNA testing please use contacts RNA testing please use contacts provided for a copy of the protocol to provided for a copy of the protocol to follow.follow.

OverviewOverview

HIV-1 RNA (NAAT) testing is important tool in HIV-1 RNA (NAAT) testing is important tool in detecting newly infected people with HIVdetecting newly infected people with HIV

HIV-1 RNA the basicsHIV-1 RNA the basics Without the pooling of specimens the ability to test Without the pooling of specimens the ability to test

for AHI would be out of reach for most Public Health for AHI would be out of reach for most Public Health DepartmentsDepartments

Current pooling methodsCurrent pooling methods Dallas County has experienced some interesting Dallas County has experienced some interesting

results with regard to the p24 band and gp160 band results with regard to the p24 band and gp160 band on Western Bloton Western Blot

ReferencesReferences1.1. Gen-Probe Aptima HIV-1 & HCV Assay Training Manual; GenProbe Gen-Probe Aptima HIV-1 & HCV Assay Training Manual; GenProbe

Incorporated, San Diego, CA.Incorporated, San Diego, CA.

2.2. Gen-Probe.2006-2009. Aptima HIV-1 RNA Qualitative Assay package Gen-Probe.2006-2009. Aptima HIV-1 RNA Qualitative Assay package insert. 501623-ART Rev. A. Gen-Probe, Inc., San Diego, CA.insert. 501623-ART Rev. A. Gen-Probe, Inc., San Diego, CA.

Acknowledgements

I would like to thank Dr. Nick Curry and Deborah Carr of Texas Department of State Health Services, Dr. Edward Bannister and Gloria Hardin of Dallas County Health and Human Services for providing me this exciting and rewarding opportunity.

Dallas County Health and Dallas County Health and Human Services Contact Human Services Contact

InformationInformationEdward R. Bannister, Ph.D.Edward R. Bannister, Ph.D.

Laboratory Services DirectorLaboratory Services Director

2377 N. Stemmons Freeway-Basement2377 N. Stemmons Freeway-Basement

Dallas, Texas 75207-2710Dallas, Texas 75207-2710

Tel: (214) 819-1952Tel: (214) 819-1952

Email: Email: [email protected]@dallascounty.org

Brian P. Emerson Brian P. Emerson

HIV-1 RNA Testing SupervisorHIV-1 RNA Testing Supervisor

2377 N. Stemmons Freeway-Basement2377 N. Stemmons Freeway-Basement

Dallas, Texas 75207-2710Dallas, Texas 75207-2710

Tel: (214) 819-1950Tel: (214) 819-1950

Email: Email: [email protected]@dallascounty.org

dallas county naat program 1

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