The current status of HCV, HIV, HBV NAT screening

Ewa Brojer Institute of Hematology and Blood Transfusion, Warsaw,

Poland

38.5 mln - population1mln donations/year

Serology - anti-HCV. anti-HIV, HBsAg

NAT - HCV RNA - since 2000

HIV RNA HBV DNA

•no anti-HBc

since 2005

Number of donationsMethod Tested HCV RNA positive Frequency

Cobas Ampliscreen48 donation

minipool3, 408, 142

491: 69,554

Chiron TMAsingle unit testing

268, 7003

1: 89,567

HCVcAgserological testing

345,0124*

1: 86.253

Total 4,021,854 56 1:71,819

HCV NAT results (March 2000 - Dec 2004)

High frequency of HCV ”window period” donors in Poland (2000-2004)

Number of donations

Tested anti-HCV(-)/ /RNA HCV(+)

USA 39 mln 170

EuropePoland excluded

50 mln 50

Poland 4 mln 56

Study of HCV „window period” donors

• anti-HCV seroconversion in all donors

• introduction of epidemiological questionaire to identify the source and date of infection

• „look back” studies of all repeat donors

Routine “Look back” samples individualy tested/ HCV RNA pos

Mini-pool NAT 26 / 1*

Single donation NAT 3 / 0

HCVcoreAg 1 / 0

*Cobas Ampliscreen positive/ Cobas Monitor negativepost-transfusion hepatitis C in the reciepient

HCV RNA detection in “ look back” samples of HCV RNA positive/anti-HCV negative repeat blood donors

• Reference Lab IHBT, Warsaw:

certification for all NAT Laboratories

• Cobas Ampliscreen (Roche):

24 donation plasma pools

• TMA HCV/HIV/HBV (Chiron):

single donations

Since 2005 - HBV, HCV and HIV NAT

BALTIC SEA

LITHUANIA

RUSSIA

BELARUS

UKRAINE

SLOVAKIA

CZECH REP.

SłupskOlsztyn

Zielona Góra

Wałbrzych

Wrocław

Łódź

Kraków

Radom

Kielce

Katowice

Opole

Szczecin

Lublin

Gdańsk

Bydgoszcz Białystok

Poznań

Kalisz

Racibórz

Rzeszów

WarszawaGERMANY

Why HBV DNA testing in pools „not larger than 24 donations”?

• Effectivity of plasma pool Cobas Ampliscreen testing revealed in preliminary study:

– 2 HBV DNA positives/60 000 dodations tested in 24 donation pools

• No preliminary studies with TMA single donation testing

• Higher sensitivity of Cobas Ampliscreen than Chiron TMA

• Lower price in tenders

HBV DNA detection in HBsAg negative blood donors

Method Tested Positives(frequency)

Discrepant*

Cobas„Pools”

182 382 21: 91 000

ChironSingle

donation

47 075 31: 16 000

9

*Discrepancies between ULTRIO and HBV Discrimination

Serological and molecular characteristics* of HBV DNA (+)/HBsAg(-) donors

Antibodies

HBc HBs HBe

HBeAg

DNAHBVcopies/ml

ALTMutation

in S region

1. Pos Neg Neg Neg 1,82x103 22 Gly145Ala

2. Pos Neg Pos Neg 1,14x102 18 No

3. Neg** Pos Pos Pos 9,55x104 19 nt

5. Pos Neg Pos Neg nt 29 nt

8. Pos Neg Pos Neg 1.26 x102 10 nt

*with participation of dr Zaaijer, dr Koppelman and dr Laperche

** HBsAg pos in PRISM test

Detected by:

mini-pool testing,single dontion testing

Examples of HBV Ultrio discrepant samples

ULTRIONo positives/ tested

Cobas isolation from:

Ultrio Discr: 850 ul 2ml*Final result Serology

3 //5 4 /10 7 /10 posPos

(~2 copies/ml)HBsAb(+)HBcAb(+)

1 / 5 1 /5 6 /10 pos Pos HBcAb(+)

1 /4 0 /3 not done posPos

(~8 copies/ml) negative

*Nuclisens Extractor (Organon Tecnika)

Examples of HBV DNA detection in Ultrio discrepant samples

ULTRIOCobas

isolation from:

No positives/ tested 850 ul 2ml*

Finalresult

Serology

Ultrio 1/ 4 0 pos 6 tested

Neg Neg Neg

Ultrio: 1/2Disc: 0/2

0 pos10 tested

Neg Neg Neg

*Nuclisens Extractor (Organon Technika)

Algorithm for HBV DNA detection in HBsAg negative donors

ULTRIO (+)

HBV Discrimination (+)ULTRIO ( +)

Discrimination (-)

Donor is

HBV DNA positive

(+) Cobas Ampliscreen

DNA from 2 ml of plasma by Nuclisens Extractor;

(-)

Repeat ULTRIO

Donor is

HBV DNAnegative

(+)

(-)