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Dallas county naat program 1

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  • 1. Dallas County Health and Human Services Laboratory Testing For Acute HIV Infection Brian Emerson HIV-1 RNA Testing Supervisor Dallas County Health and Human Services Laboratory Ph: (214)-819-1950 Email: brian.emerson@dallascounty.org
  • 2. Discussion Topics
    • Introduction to HIV RNA NAAT: the basics
    • Dallas County HIV Algorithm
    • Pooling Methods
    • Dallas County Laboratory Experience
  • 3. Introduction to HIV RNA Testing
    • Dallas County Laboratory utilizes the Aptima HIV-1 RNA Qualitative Assay (manufactured by Gen-Probe Inc.) for detection of acute HIV infection (AHI).
    • Aptima HIV-1 RNA Qualitative Assay is approved by the FDA for a diagnostic test and can be used in place of Western Blot.
  • 4. Introduction to HIV RNA Testing
    • Aptima HIV-1 RNA Assay is an in vitro nucleic acid-based amplification test (NAAT) used for the detection of HIV-1 RNA in human plasma and serum
    • The presence of HIV-1 RNA in the plasma or serum of a patient without antibodies to HIV-1 is an indication of acute HIV-1 infection
  • 5. Introduction to HIV RNA Testing
    • The NAAT assay contains four main sections:
      • Target Capture – RNA of virus
      • TMA (Transcription-Mediated Amplification)
      • HPA (Hybridization Protection Assay)
      • DKA (Dual Kinetic Assay)
    Gen-Probe Aptima HIV-1 & HCV Assay Training Manual
  • 6. Introduction to HIV RNA Testing
    • The RNA is isolated from the specimen via the use of target capture, which helps reduce false negatives by removing inhibitors. A detergent is used to solubilize the viral envelope, denature proteins, and release the viral genomic RNA.
    • Specific regions of the HIV-1 will be hybridized to the HIV-1 RNA target, if present, in the test specimen
    • The hybridized target is captured onto magnetic micro particles that are separated from the serum in a magnetic field. All other extraneous components from the reaction tube will be washed away during the wash cycle.
  • 7. Introduction to HIV RNA Testing
    • TMA is a transcription-based nucleic acid amplification method that utilizes two enzymes
    • Amplification of nucleic acid target producing amplified RNA product (amplicon)
      • RNA  DNA  RNA
    • Combined with HPA detection in a single tube format
    • Hybridization of DNA probe with amplicon
    • Degradation of label on unbound probe
    • Detection of chemiluminescent label on DNA probe
      • Measured in Relative Light Unit (RLU)
  • 8. Introduction to HIV RNA Testing Gen-Probe Aptima HIV-1 & HCV Assay Training Manual Gen-Probe Aptima HIV-1 & HCV Assay Training Manual Negative Specimen Positive Specimen
  • 9. Dallas County HIV Algorithm Bio-Rad Multispot HIV-1/HIV-2 Rapid Test Blood 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 Repeat HIV testing in 1 month AHI Protocol NAAT Negative HIV – (Retest based on risk factors)
  • 10. Dallas County HIV Algorithm Blood sample received for HIV test ELISA or EIA is performed EIA Negative Pooled NAAT Testing NAAT Positive AHI Protocol NAAT Negative HIV Negative (Retest based on risk factors)
  • 11. Dallas County HIV Algorithm Bio-Rad Multispot HIV-1/HIV-2 Rapid Test Blood sample received for HIV test ELISA or EIA is performed EIA + (Test repeated X2) Western Blot Western Blot Positive Western Blot Indeterminate HIV Positive NAAT Testing NAAT Positive NAAT Negative AHI Protocol Repeat HIV testing in 1 month
  • 12. Dallas County HIV Algorithm Bio-Rad Multispot HIV-1/HIV-2 Rapid Test Blood sample received for HIV test ELISA or EIA is performed EIA + (Test repeated X2) Western Blot Western Blot Negative NAAT Testing NAAT Positive NAAT Negative AHI Protocol Repeat HIV testing in 1 month
  • 13. Pooling Methods
    • The ability to combine specimens into pools greatly reduces the cost for detection of acute HIV infection.
    • Pooling specimens will reduce the cost by 90%. (using 30,000 specimen per year for analysis)
    • Pooling is not part of the package insert. The pooling method was verified and validated within the Dallas County Health Laboratory.
    • The pooling procedure is performed manually
  • 14. Pooling Methods
    • The specimens requesting HIV testing are first tested using either the BioRad HIV -1/2 Plus O EIA assay or Clearview Rapid HIV method (used in the STAT lab and Mobile Vehicle).
    • The specimens are separated into High Risk or Low Risk category
    • High or Low Risk is determined by the patient risk factors and submitter.
    • (Ex. STD Clinic = High Risk, Family Planning Clinic = Low Risk)
  • 15. Pooling Methods
    • Currently utilizing pools of 10 for all high risk EIA and Rapid Nonreactive (NR) specimens.
      • Pooling is performed same day as the EIA
      • Nonreactive results are available 1 day after draw date
      • Reactive results are available 3 days after draw date
    • Testing only High Risk specimens due to cost
    • Western Blot Nonreactive and Indeterminate and AHI suspects are tested individually.
  • 16. Pooling Methods
    • 10 individual specimens are assigned to a specific pool number
    • 100 μ l of serum is pipetted into labeled pool tube
    • Pool of 10 individual specimen
    100 μ l of serum removed Pool Tube 1.0ml of serum
  • 17. Pooling Methods
    • Example specific pool number : High-042110-1001 : High = High Risk Specimens, 042110= date, Pool # to date
    • If specific pool result is nonreactive then all specimens assigned to the pool are reported as nonreactive.
    • A nonreactive result means no HIV-1 RNA detected in pool tube
  • 18. Pooling Methods
    • If pool result is reactive then the pool will be broken back down to 10 individual specimens and tested individually
    • Once the reactive specimen is determined, one more individual test will be performed before the specimen will be reported as an acute infection
    • The result of the specimen being reported as an acute infection will be given to the Medical Director (STD Clinic), DIS Manager, and Aids Surveillance
  • 19. Pooling Methods
    • Limit of Detection with a pool of 10 is 300 copies of HIV-1 RNA per ml
    • This means the specimen must have 300 copies per milliliter for the assay to detect the presence of HIV-1 RNA
    • Limit of Detection for testing an individual sample is 30 copies per milliliter
  • 20. Dallas County Laboratory Experience
  • 21. Dallas County Laboratory Experience
  • 22. Dallas County Laboratory Experience Reginald Karen
  • 23. Dallas County Laboratory Experience Daniel Serinaldi Shayma Haq
  • 24. Dallas County Laboratory Experience
    • Dallas County Health Laboratory experienced a 44% increase in patients requesting a HIV test from 2007 to 2009
    • Dallas County 2010 projection will be over 60,000 specimens for HIV testing alone
  • 25. Dallas County Laboratory Experience
    • The HIV RNA program went live in July 2009
    • From July 2009 to April 2010 a total of 30,617 High-Risk specimens tested
    • 26 Acute infections were discovered
      • 24 in Dallas County and surrounding area
      • 2 from outside Dallas County
  • 26. Dallas County Laboratory Experience
    • 19% (5/26) of the AHI tested positive for Syphilis
    • 15% (4/26) of the AHI tested positive for other STDs excluding Syphilis (ex. Gonorrhea)
    • 35% (9/26) of the AHI did not test positive for any other STDs besides HIV
    • 31% (8/26) unknown if any other STDs were present
  • 27. Dallas County Laboratory Experience * Thru April 1, 2010 ŧ Western Blot Indeterminate specimens not included 6.9 12 174 21108 2010* 1.5 9 581 57060 2009 % Increase Confirmed by HIV-1 RNA ŧ Confirmed by Western Blot Total Specimen Tested for HIV Year
  • 28. Dallas County Laboratory Experience
    • Acute HIV-1 Infections
    • 21 were BioRad HIV ½ Plus O EIA or Clearview Rapid HIV Nonreactive and Western Blot Nonreactive (No Bands Present)
    • 5 were Western Blot Indeterminate
      • 1 with only p24
      • 1 with p24 and p51/p55
      • 3 with gp160
  • 29. Dallas County Laboratory Experience 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 )
  • 30. Dallas County Laboratory Experience
    • HIV-1 RNA (NAAT): Nonreactive
    • EIA Assay: Reactive
    • Nonreactive and Indeterminate Western Blots
      • Western Blot:
      • 63% (22/35) were Nonreactive for Western Blot (No Bands Present)
      • 34% (12/35) were Indeterminate with only the p24 band
      • 3% (1/35) were Indeterminate with only the gp160 band
    • Interesting once the p24 band will be used for AHI detection in the 4 th Generation EIA
  • 31. Dallas County Laboratory Experience
    • Some Interesting Cases:
    • Blood drawn on 03/17/10 and again on 03/23/10
    FIRST BAND TO APPEAR ON THE WESTERN BLOT: gp160 HIV-1 RNA: R HIV-1 RNA: R gp160 No Bands Present Western Blot: Indeterminate Western Blot: NR BioRad EIA: R Clearview Rapid: NR 03/23/10 03/17/10
  • 32. Dallas County Laboratory Experience Some Interesting Cases: Blood drawn on 01/05/10 and again on 01/15/10 HIV-1 RNA: R HIV-1 RNA: R gp160, p24 Bands Present No Bands Present Western Blot: R Western Blot: NR BioRad EIA: R BioRad EIA: NR 01/15/10 01/05/10
  • 33. Dallas County Laboratory Experience Interesting Case:
      • Possible reason: If patient is already on antiretroviral medication then the viral load will be below the detectable range.
      • The patient’s history is now being investigated for a possible explanation for the HIV-1 RNA test result.
    NR NR HIV-1 RNA (Dallas) HIV-1 RNA (Houston) All bands present Bands Present R Western Blot R (x2) BioRad EIA 04/08/10 Blood Drawn
  • 34. Dallas County Laboratory Experience 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.
    HIV-1 RNA: NR HIV-1 RNA: R No Bands Present No Bands Present Western Blot: NR Western Blot: NR Clearview Rapid: NR BioRad EIA: NR Clearview Rapid: NR BioRad EIA: NR 02/04/10 01/21/10
  • 35. Dallas County Laboratory Experience
    • Dallas County Health and Human Services Laboratory tests Nonreactive and/or indeterminate Western Blots for other Public Health Departments throughout Texas.
    • If anyone is interested in sending Dallas County Laboratory specimens for HIV-1 RNA testing please use contacts provided for a copy of the protocol to follow.
  • 36. Overview
    • HIV-1 RNA (NAAT) testing is important tool in detecting newly infected people with HIV
    • HIV-1 RNA the basics
    • Without the pooling of specimens the ability to test for AHI would be out of reach for most Public Health Departments
    • Current pooling methods
    • Dallas County has experienced some interesting results with regard to the p24 band and gp160 band on Western Blot
  • 37. References
    • Gen-Probe Aptima HIV-1 & HCV Assay Training Manual; GenProbe Incorporated, San Diego, CA.
    • Gen-Probe.2006-2009. Aptima HIV-1 RNA Qualitative Assay package 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.
  • 38. Dallas County Health and Human Services Contact Information
    • Edward R. Bannister, Ph.D.
    • Laboratory Services Director
    • 2377 N. Stemmons Freeway-Basement
    • Dallas, Texas 75207-2710
    • Tel: (214) 819-1952
    • Email: [email_address]
    • Brian P. Emerson
    • HIV-1 RNA Testing Supervisor
    • 2377 N. Stemmons Freeway-Basement
    • Dallas, Texas 75207-2710
    • Tel: (214) 819-1950
    • Email: [email_address]