Transcript HIV GENETIC DIVERSITY - 2012 HIV Diagnostics Conference

Utilizing a Non-Commercial RealTime 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
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HIV antibody (-) sera in the Public Health
Laboratory setting
Overview of the validation of an in-house realtime 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: HIV1/HIV-2 Selective Synthetic
Peptide EIA and HIV-1 p24
Ag EIA
• Small numbers of HIV-1 seroconversions 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 crosscontamination 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
16
15
Positive ( cT.< 38.00)
14
Negative (cT.>38.00)
# of Replicates Tested
12
9
10
7
8
6
6
4
Avg. (cT.)
29.29
Avg.( cT.)
32.04
Avg. (cT.)
32.74
2
2
1
4
Avg.(cT.)
34.37
0
0
2500
2000
1500
HIV-1 RNA copies/ml of One HIV-1(+) Serum in pool of 20 Members
1000
Testing of specimens with known
HIV-1Viral Loads
HIV-1 Viral Load
bDNA (RNA
copies/ml)
<75 (Below Limit of
detection)
>495 - <2,000
>2,000-10,000
>10,000-,50,000
>50,000-<500,000
>500,000
HIV-1(gag)Real- Mean(ct.) Realtime PCR(+)
time PCR of
/ # tested
Positives
0/80
3/10
10/13*
8/8
10/10
3/3
N/A
30.20
31.00
28.71
25.36
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 BBI Roche
First PCR HIV-1 RT(rt) HIV-1 Interpretation of Real-time
Bleed Viral Load Gag PCR(ct)
PCR Results
0
70,000
34.04
HIV-1 RNA Detected
7
>800,000
22.90
HIV-1 RNA Detected
11
300,000
25.39
HIV-1 RNA Detected
15
10,000
33.26
HIV-1 RNA Detected
18
2,000
38.63
HIV-1 RNA Detected
22
2,000
>45
Not Detected
25
1,000
>45
Not Detected
29
1,000
>45
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 46hr.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 Study
HIV-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 genotyping 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
Testing Period
2004: 4th Q.
2004: 3rd Q.
2004: 2nd Q.
25,000
2004: 1st Q.
2003: 4th Q.
2003: 3rd Q.
2003: 2nd Q.
2003: 1st Q.
2002: 4th Q.
2002: 3rd Q.
2002: 2nd Q.
2002: 1st Q.
2001: 4th Q.
2001: 3rd Q.
2001: 2nd Q.
2001: 1st Q.
2000: 4th Q.
2000: 3rd Q.
2000: 2nd Q.
2000: 1st Q.
1999: 4th Q.
1999: 3rd Q.
1999: 2nd Q.
1999: 1st Q.
1998: 4th Q.
1998: 3rd Q.
1998: 2nd Q.
1998: 1st Q.
1997: 4th Q.
Total # Specimens Tested
Maryland DHMH Laboratory: Quarterly HIV Antibody Testing Wokloads
1997-2004
Oral Fluids
Blood
20,000
15,000
10,000
5,000
0
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 noncommercial 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