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)”

• 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: ??

“………………..”??

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).

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

HCV

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.

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Source: Hepatology 2000;31:777-82; Hepatology 1997;26:62S-65S;CDC, unpublished data

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

Questions

• Is there a significant change or decrease in HCV sero-prevalence among transplant donors?

• What factors are associated with HCV positivity?

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)

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

%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

%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

%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

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

“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

HIV-1

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.

Practical aspects of NAT

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

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.

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.

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)

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

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

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

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

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

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

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!

Thank you for your attention!