national institute of transplantation experience with nat testing
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National Institute of Transplantation experience with NAT testing. Marek Nowicki, PhD , Scientific Director National Institute of Transplantation, Los Angeles. Important dates…. “Hepatitis C Virus Transmission from an Antibody-Negative Organ and Tissue Donor (MMWR, 2003)” - PowerPoint PPT PresentationTRANSCRIPT
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National Institute of Transplantation experience with NAT testing
Marek Nowicki, PhD, Scientific Director
National Institute of Transplantation,Los Angeles
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Important dates….“Hepatitis C Virus Transmission from an Antibody-Negative
Organ and Tissue Donor (MMWR, 2003)”
• 2004 AOPO Meeting, New Orleans: NAT is recommended
“Four Transplant Recipients Contract HIV, Hepatitis C From High-Risk Organ Donor (2007)”
• 2008 AOPO Meeting, Park City Utah: ??
“………………..”??
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Why nucleic acids testing? • The donor has negative laboratory test results during the early
stages of infection, known as the window period (HIV and HCV).
• The existence of a chronic carrier state in which a clinically asymptomatic donor will persistently test negative for antibody (HCV).
• Certain viruses have a large degree of genetic diversity and laboratory may fail to identify donors infected with a particular atypical genetic variant (HIV and HCV).
• Laboratory error in performing screening tests; however, the occurrence of such errors is thought to be extremely rare (HIV
and HCV).
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Declining time to detection of HBV, HCV, HIV markers during the window phase following infection
Infection
HCV RNA HCV Ag
EIA 3.0
EIA 2.0
EIA 1.0
0 13 14 70 80 150 (days)
Infection
HIV RNA
p24 Ag
EIA 3.0
0 11 16 22 (days)
HCVHCV
HIVHIV
Infection
HBV DNA
EIA 3.0
0 41 56
HBVHBV
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HCV
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Background
• After successful introduction of screening for antibodies to the hepatitis C virus (HCV) in early 90’s, and more recently, also for HCV RNA, several authors reported a dramatic decrease of new acute HCV infections in the US (Hepatology 2000;31:777-82; Hepatology 1997;26:62S-65S; CDC)
• Others published predictions of HCV epidemic trajectories, showing steady decline of the number of infected individuals in the next 30 years (Davis, LT, 2003)
• Since ‘02 our laboratory has been evaluating HCV serostatus of approx 850 cadaveric organ donors/year. Recently (’05) our testing algorithm was supplemented by the sensitive TMA-based NAT assay detecting HCV RNA.
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Estimated Incidence of Acute HCV Infection, United States,
1960-2001
0
20
40
60
80
100
120
140
1960 1965 1970 1975 1980 1985 1989 1992 1995 1998 2001
Year
New
Infe
ctio
ns/1
00,0
00
Decline intransfusion recipients
Decline in injection drug users
Source: Hepatology 2000;31:777-82; Hepatology 1997;26:62S-65S;CDC, unpublished data
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Prevalence of HCV Infection by Age and Gender, United States
0
1
2
3
4
5
6
6-11 12-19 20-29 30-39 40-49 50-59 60-69 70+
Age in Years
% H
CV
+
Males
Females
Total
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Questions
• Is there a significant change or decrease in HCV sero-prevalence among transplant donors?
• What factors are associated with HCV positivity?
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HCV seroprevalence – 3 CA OPOs
0
2
4
6
8
10
12
2002 2003 2004 2005 2006
HC
V%% OPO 3 (S. Cal)
OPO 1 (N. Cal)
OPO 2 (N. Cal)
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Material and Methods
• We tested 4,032 consecutive donors from 3 CA OPOs (2 from N. Cal and 1 from S. Cal)
• Testing period: 2002-2007
– EIA (Ortho), confirmed with RIBA (Chiron)
– After 2005 HCV RNA NAT (Procleix, Chiron)
• Statistical Methods
– Chi-square and logistic regression
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%HCV+ Donors in 3 CA OPOs
0
5
10
15
20
OPO 1 OPO 2 OPO 3Time Period
%02-0304-0506-07
Increase in OPO1, trend not significant in OPO 2 & 3
Year
P=0.01 P=NS P=NS
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%HCV+ Donors by Donor Age
02468
101214161820
0-20 21-30 31-40 41-50 51-60 61-70 >70Donor Age
%OPO 1OPO 2
>80% HCV donors were between age 41-60
P<0.001 for both OPOs
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%HCV+ Donors with other Serology
0
5
10
15
20
25
30
HIV HBsAb HBcAb EBV CMV
%HCV++
NegPos
P<0.001P=0.001
N=21 N=218 N=467 N=1169 N=3319
P=0.048 P=0.012
P<0.001
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Factors Associated with HCV
Factor Reference Odds Ratio, 95% CI P
2004-05 2002-03 1.61, 0.97-2.69 0.065
2006-07 2.03, 1.26-3.30 0.004
Age 41-60 <40 or >60 1.77, 1.20-2.61 0.004
HBcAb+ HBcAb- 9.06, 6.17-13.3 <0.001
• Prevalence of HCV+ increased• HCV is almost twice as likely in 41-60 age group• 9 times more likely when HBcAb positive
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“Take home Message”
• HCV+ rates differ among CA OPOs
• Rates were highest for age 41-60
• Rates increased in 2006-2007 time period
– Change in targeted donor population?
– More stringent testing (NAT)?
– Increased use of non-optimal donors?
• Further studies are needed to examine factors associated with outcomes for HCV+ donors
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HIV-1
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CDC Holds New Annual HIV Infection Estimates in Abeyance Till Next Year
• ATLANTA, Dec. 3: annual U.S. HIV infection rate maybe 50% higher than suspected, the CDC said the new estimate remains tentative.
• The new methodology would boost the estimate of new HIV infections from 40,000 per year to more than 60,000 (K. Fenton, M.D., Ph.D., director of the National Center for HIV/AIDS, Viral Hepatitis, STD, and TB Prevention)
• Validated estimates will be issued early in 2008, said the CDC.
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Practical aspects of NAT
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Why TMA/Procleix?
• Specifically developed for donor screening under NIH (1996, NLHBI) contract
• First licensed NAT for HIV/HCV
• Robust and well suited for time sensitive applications:
Minimal sample preparation Internal Control (IC) added to each reaction tube
Entire assay performed in one tube
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Roche COBAS AMPLISCREEN v. Chiron Procleix
Ampliscreen - Ampliscreen - 5 steps5 steps*
• Specimen preparation (ultra-centrifugation)
• Reverse transcription of RNA to generate (cDNA)
• Polymerase chain reaction** (PCR) amplification of cDNA
• Hybridization of the amplified products to probes specific to the target
• Detection of the probe-bound products by colorimetric determination
• The entire test process takes approximately 6 hours.
*After the specimen preparation is complete, the remaining processes are fully automated on the COBAS instrument.
**The reverse transcription and PCR amplification of viral target and internal control
target occur simultaneously.
Procleix - Procleix - 3 steps3 steps
• Specimen preparation (target capture with detergent +magnetic microparticles)
• Transcription Mediated Amplification (TMA) to amplify HIV-1/HCV target RNA
• Detection by using a chemiluminescent probe and luminometer *
• The test process takes approximately 3.5 hours.
*An internal control is added to each reaction to control for all steps of the process. If the HIV-1/HCV test is reactive, discriminatory tests are used to differentiate between the two viruses. These tests use the same process as the multiplex assay.
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Procleix validation - Low Viral Load Specimens
• 4 HCV RNA negative blood specimens were spiked with the Hepatitis C Virus (end concetration approx. 130 copies/ml).
• Tested using the Chiron Procleix HIV-1/HCV Assay and the Roche Diagnostics AMPLICOR HCV Test, version 2.0.
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Results
Day Tested % Of Samples that Tested Reactive/Positive
Procleix HIV-1/HCV Assay AMPLICOR Assay
Day 1 100% (4/4) 100% (1/1)
Day 2 100% (4/4) 50% (4/4)
Day 3 100% (3/3) 50% (2/4)
Day 4 75% (3/4) 50% (2/4)
Day 5 100% (4/4) 50% (2/4)
Day 6 75% (3/4) 25% (1/4)
Day 7 100% (4/4) 75% (3/4)
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The longitudinal performance of EQC specimens
0.00
10.00
20.00
30.00
9/1 9/15 9/29 10/13 10/27 11/10 11/24
S/C
O r
atio
mean ± SD signal to cutoff (S/CO) ratios:
HIV 18.7 ± 6.6
HCV 9.5 ± 5.0
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NAT Routine QA and contamination prevention – NIT experience
• All technician trained by the manufacturer or manufacturer’s approved trainer, regular retraining
• Laboratory area arranged with a uni-directional workflow
• Negative air pressure in the NAT lab’s rooms
• Monthly swabbing to identify possible contaminated areas before they become a major problem in the lab and spread into other areas
• Randomly placed negative specimens in the reaction racks
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Procleix HIV-1/HCV Assay Protocol
Thaw Reagents(RT)
Reagent AliquotSample PipettingwTCR Pipetting
RNA Release incubation(60°C)
30 min 20±1 min
Solid Phase Capture(RT)
14-20 min
Target Capture Process (TCS)
9-20 min
1st Wash
4-10 min
2st Wash
4-10 min
Amp I(60°C)
Amp II(41.5°C)
10±1 min9-20 min
Amp III (41.5°C)
60±5 min
HPA(60°C)
15±1 min
Selection(60°C)
10±1 min
Cooling Step(RT)
10 min
READ
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Logistics of NAT Testing
• All specimens are tested individually unless arrived within 2 hrs of each other – NO batching for most specimens
• Run starts as soon as the lab is notified about incoming specimens – time savings of ~ 0.5-1 hr
• All specimens are tested undiluted and diluted - to prevent NAT inhibitors
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Summary of NAT testing at the NIT
Laboratory since 9/2004 • We performed 2943 Procleix runs testing 3655
donors
• 0.8% gave false positives NAT, all resolved before reporting
• 4.13% of donors were HCV RNA+
• 0.24% of donors were HIV RNA+
• 0.19% of donors were HCV+HIV RNA positive
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Conclusions• NAT is doable in OPO setting!
• Doesn’t affect Lab turnaround time
• It provides additional layer of safety for organ transplant recipients
• Identifies viremic donors
• Contributes to EIA testing confirmation
• Doesn’t result in excessive organ donor rejection
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Why OPOs should run NAT even if is not required at this time?
• To prevent “window” HCV and HIV donations
• To detect “silent” or cryptic HIV/HCV carriers
• “State of the art” in donor testing = the best we can do to prevent transmission
• Because Blood Banks are doing it!
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Thank you for your attention!