Utilizing a Non-Commercial Real-Time PCR to Detect HIV-1 RNA in HIV Antibody Negative Diagnostic Sera Submitted to The Maryland

Public Health Laboratory

Robert A. Myers Ph.D.

Maryland DHMH Laboratories

Presentation Overview• Reasons for performing HIV RNA NAAT of

HIV antibody (-) sera in the Public Health Laboratory setting

• Overview of the validation of an in-house real-time reverse transcribed PCR for HIV-1 NAAT testing

• Review of NAAT testing pilot study data to date • Possible reasons for not yet detecting HIV-1

RNA (+) Antibody negative specimens in our testing population

• Conclusions and recommendations

Reasons for performing HIV NAAT on HIV Antibody(-) Specimens in a Public Health Laboratory Setting

• Shorten “window period” of HIV sero-conversion and improve the diagnostic capabilities of the laboratory

• Higher prevalence of HIV sero-converting patients in the “high risk” populations tested by public health laboratories

• Possibly reducing HIV transmission rates by quickly identifying antibody(-) viremic individuals

• Possibly utilizing the data to guide epidemiological investigations and intervention strategies

Supplemental HIV Serology Testing to Identify HIV Seroconversion

• Since the late 1980’s the MD DHMH lab routinely conducts supplemental tests to identify possible HIV sero-conversion from all ELISA(+) diagnostic specimens that are not confirmed by W.B.

• Supplemental tests: HIV-1/HIV-2 Selective Synthetic Peptide EIA and HIV-1 p24 Ag EIA

• Small numbers of HIV-1 sero-conversions and HIV-2 positive patients have been consistently identified using this approach

Pilot NAAT Study: In-house HIV-1 Real-time PCR Assay

• For cost effectiveness use existing laboratory infrastructure: instrumentation and molecular diagnostics staff

• Real Time PCR technology is used by most public health labs for molecular diagnostics(i.e., WNV, BT testing )

• Modify published Real-time PCR based HIV-1 gag assay based on Taqman chemistry

Reference: [J.Clinical Micro.Vol.39(12) 4302-4308. 2001]

Real-time PCR Basics

• Simultaneous amplification and detection of agent specific nucleic acid sequences

• Detection amplified PCR products by the specific hybridization of sequence complimentary DNA probes

Advantages of Real-time PCR Assays

• High through-put• Reduces cross-

contamination via PCR products (amplicons)

• Sensitivity: low levels of target molecules (i.e.viral gene sequences) can be detected

HIV-1 gag Taqman Real-time Developmental PCR

• 100ul/ sample (serum/plasma) tested in pools of 20 samples

• Concentrate HIV-1 particles in each pool by high speed centrifugation: 1hr.@25,000Xg

• Column based manual RNA extraction of each pellet

• One-step reversed transcribed real-time PCR using 2 gag primer sets and 1 fluorescent labeled probe

HIV-1 gag Taqman Real-time Developmental PCR: Controls

• RNA extraction controls(2): (-) sample buffer(BA) and LaCrosse virus lystate

• Internal positive control (IPC) is added and run with each extracted pool to determine if PCR inhibitors are present

• HIV-1 RNA assay low positive sensitivity and

NTC(-) controls

HIV-1 gag Taqman Real-time Developmental PCR: Data

• Fluorescent signals emitted indicative of target amplification and probe binding

• Positive defined by PCR cycle # when the fluorescent signal crosses the back-ground threshold; termed crossing threshold (cT.)

• Break down PCR(+) pools and run real-time PCR on individual samples; confirm individual HIV-1 RNA(+) samples with bDNA quantitative assay[(pol) target]

Real-time RNA HIV-1 gag PCR: Reproducibility /Sensitivity Validation

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HIV-1 RNA copies/ml of One HIV-1(+) Serum in pool of 20 Members

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Positive ( cT.< 38.00)

Negative (cT.>38.00)

Avg. (cT.) 29.29

Avg.( cT.) 32.04

Avg. (cT.) 32.74

Avg.(cT.) 34.37

Testing of specimens with known HIV-1Viral Loads

HIV-1 Viral Load bDNA (RNA copies/ml)

HIV-1(gag)Real-time PCR(+)

/ # tested

Mean(ct.) Real-time PCR of

Positives<75 (Below Limit of

detection) 0/80 N/A>495 - <2,000 3/10 30.20>2,000-10,000 10/13* 31.00

>10,000-,50,000 8/8 28.71>50,000-<500,000 10/10 25.36

>500,000 3/3 22.66

* Note: One negative plasma genotyped as HIV-1 Subtype:C

Evaluation of the Biological Sensitivity of Real-time RNA HIV-1 (gag)

BBI Sero-Conversion Panel:PR940

Date Since First Bleed

BBI Roche PCR HIV-1 Viral Load

RT(rt) HIV-1 Gag PCR(ct)

0 70,000 34.047 >800,000 22.90

11 300,000 25.3915 10,000 33.2618 2,000 38.6322 2,000 >4525 1,000 >4529 1,000 >45

HIV-1 RNA Detected

Interpretation of Real-time PCR Results

HIV-1 RNA Detected HIV-1 RNA Detected HIV-1 RNA Detected

HIV-1 RNA Detected Not Detected Not Detected Not Detected

Cost Analysis: HIV-1 Real-time RNA Assay

• RNA isolation /pool : $6.80• Real-time PCR reagents/pool: $5.11• Miscellaneous lab supplies( tips,tubes etc.)

/pool: $2.65• Cost of controls(4/run )/avg.# pools/run $1.58• Total cost/pool $16.14 (avg. 20

specimens/pool)[ includes control costs]• Cost / HIV antibody (-) specimen $0.81

Can sera from routine HIV antibody testing be used for molecular based testing?

• Dogma: plasma specimens are required for molecular testing (separated from RBC’s 4-6hr.post collection)

• Explore the possibility of using the plasma sample for both serological and molecular diagnostic (NAAT) HIV testing and HIV genotyping of positive samples

• Plan to provide centrifuges to large volume clinics to process plasma separator tubes(PPT)

Serum vs. Plasma StudyHIV-1 Drug Resistance Genotyping PCR

• Determine the suitability of using routinely handled HIV(Ab) diagnostic serum specimens vs. ideally collected plasma samples for molecular based HIV testing

• 54 HIV-1 antibody(+) serum/ plasma pairs were collected

• HIV-1 viral loads (bDNA) established from the ideal plasma sample from each serum/plasma pair

• HIV-1 Drug Resistance geno-typing PCR was performed on each sample

Pilot Study:Serum vs. Plasma HIV-1 Drug Resistance Genotyping PCR

• No differences in serum/ plasma samples for HIV-1 PCR amplifications for HIV-1 genotyping ( HIV-1 viral loads as low as 700 copies/ml could be amplified equally in both serum and plasma)

• PCR genotyping non-amplifiable samples were equal for serum or plasma (most viral loads <75 copies/ml)

• Conclusion : Diagnostic sera submitted for routine HIV antibody testing are suitable for molecular testing

Initial Results of Real-time PCR Testing of HIV Antibody(-) Diagnostic Sera• Over 816 pools representing over 15,000 specimens have

been tested from Oct. 6, 2004 to Feb. 6, 2005• Pools are prepared daily 2-3 days after HIV serology

testing is complete. Real-time PCR testing is performed 2X/week

• To date no HIV-1 RNA(+) specimens have been detected• Blinded HIV-1 RNA (+) challenge samples were

successfully detected as part of an internal QA program (challenge specimens HIV-1V.L. approx. 10,000-9,000 copies/ml)

Possible Reasons for Not Detecting HIV-1 RNA(+)/Antibody(-) Specimens

1.) Limited number of specimens tested to date• Prevalence of HIV-1 RNA(+)/Ab(-) samples

from acutely infected individual is probably less than 1/15,000

• Goal of this pilot study is to test 100,000 to 150,000 HIV antibody negative specimens before drawing any conclusions about the utility of HIV-1 NAAT testing

Possible Reasons for Not Detecting HIV-1 RNA(+)/Antibody(-) Specimens

2.) Routine use of a more sensitive HIV-1/HIV-2 synthetic peptide based screening ELISA may detect more early sero-converting patients that are not detected by more commonly used viral-lysate HIV-1 screening assays• The MD DHMH screens all blood samples in both HIV-

1/HIV-2 synthetic peptide and HIV-1 viral-lystate EIAs• A review of DHMH testing records from 2003 & 2004:

>111,500 blood specimens dually tested, found only one sample that tested HIV-1 Ab peptide(+)/v.l.(-) and was subsequently identified as a HIV-1 sero-converter by confirming the presence of HIV-1 p24 Ag in the sample

Possible Reasons for Not Detecting HIV-1 RNA(+)/Antibody(-) Specimens

3.) Shift in testing high-risk patients from blood based HIV antibody assays into oral fluid or rapid HIV testing technologies • Oral fluid testing is widely recognized as a useful tool to

obtain specimens from recalcitrant clients in unconventional settings

• Many sites are adopting oral fluid testing for convenience rather than the inability to collect a blood sample for a client

• Over 25% of the HIV diagnostic specimens sent to our lab in 2004 were oral fluid specimens

Maryland DHMH Laboratory: Quarterly HIV Antibody Testing Wokloads 1997-2004

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Oral FluidsBlood

Popularity and Pitfalls of Oral Fluid (OF) HIV-1 Antibody Testing

• “Reduced sensitivity and specificity of testing with Orasure HIV-1 specimens compared with blood specimens.” (From Oral Fluid Vironostika HIV-1 ELISA kit insert)

• OF testing has greatly reduced the number of high risk patients that can be tested by NAAT in our lab (i.e 7,200 oral fluid tests/year from prison inmates )

The Impact of HIV Rapid Testing on NAAT of HIV Antibody (-) Specimens• Rapid testing is an increasingly popular tool for high risk clients

in unconventional settings; over 5800 tests performed in Maryland in 2004.

• Sensitivity of HIV rapid tests is better in comparison to the the conventional OF EIA. HIV seroconversion specimens may be recognized rapid tests and confirmed by follow-up blood testing.

• Problems can occur when OF tests are used to confirm rapid test positives due to the relative insensitivities of OF HIV tests (EIA and WB).

• Blood spot testing may provide an alternative sample to whole blood for NAAT of HIV antibody negative specimens. (Problems with RNA stability on blood spots?)

Possible Insensitivities of the Real-time RNA PCR to detect gag variant non-B

subtype HIV-1 viruses?4.) Genetic Variation of the gag primer/probe target sequences

• When evaluating HIV-1 bDNA RNA(+) plasmas to establish a linearity curve to quantify viral loads of serum specimens by Real-time PCR that could not be amplified from drug resistance genotyping, we discovered several plasmas (5) with high viral loads (12,952 to >500,000) that could not be amplified in the real-time PCR.

• Attempts to sequence the gag region corresponding to the primer probe binding sites are in progress.

• Inability to amplify HIV-1 strains with divergent sequences in Real-time RNA HIV-1 PCR gag target regions could reduce the utility of this assay.

Concluding Remarks• We have modified a published reverse transcribed

real-time HIV-1 gag PCR to test pools of HIV antibody(-) sera for acute RNA(+)/antibody (-)HIV-1 infections.

• The assay reproducibly detects viral loads at a minimum of 2,000-2,500 copies/ml / individual sample in a pool of 20 samples.

• The assay is cost effective (cost est. < $ 0.85 /sample).

• To date over 15,000 HIV antibody(-) samples have been tested and no HIV-1 RNA(+) patients have been detected.

Concluding Remarks• The continued trend of using point of service

and/or non-blood based testing technologies could significantly reduce the number of high risk acutely HIV infected individuals that are submitted to public health labs for traditional HIV antibody testing and thus limit the usefulness of HIV NAAT of antibody(-) samples.

• The utility of continuing to perform this non-commercial HIV-1 real-time RNA PCR assay to detect HIV acute infections in our laboratory remains to be determined.

Acknowledgements:

• The staff of Maryland DHMH Retrovirology, Molecular Diagnostics, and Molecular Epidemiology Laboratories

• The staff of Maryland DHMH AIDS Administration

• The organizers of the APHL meeting and

Dr. Branson of the CDC