Virus Detection by NATNew and continuous challenges

IPFA / PEI 18th international workshop on„Surveillance and screening of blood borne pathogens“

Dublin, May 2011

W. Kurt Roth, MD CEO

Challenges for NAT Testing

• Sensitivity• Throughput• Flexibility• Viruses

– „Old“ portfolio viruses• HCV• HBV• HIV-1

– „New“ portfolio viruses• Human Cytomegalo Virus• West Nile Virus• Chikungunya Virus• Dengue

Scalability:Low to high throughputFittable to smaller lab spaces Test sensitivity adjustable via pool size

Flexibility:Processing of different sample types on the same platformPooling on the same platform or separatelyOpen to integrate new tests without changing the platform

Safety:

Single-use reagentsAll reagents monitored via barcodes

Reagent requirements cross-referencing with work list Samples tracked and monitored via barcodes

CE mark

Performance:High sensitivity / high specificityHigh throughputBroad spectrum of viruses

User friendlyWalk-away system

autoX-System Design Guidelines

HCV Transmissions in Germany

0

2

4

6

8

10

12

14

16

1990 91 92 93 94 95 96 97 98 99 2000 1 2 3 4 5 6 2007

HCV transmissions HCV NAT-only positive

HCV NAT mandated

M. Nübling et al. Transfusion, 2009

HBV NAT Window Periods and Course of Infectivity

D

Infection infectiousPCR

negativedetection

limitHBV DNA

peakdetection

limitPCR

negative infectious cure

≥ 1 copy/circulation

vol. (5L)

≥ 1 copy/transfusion

400 ml

1 copy /ml 60copies/ml

60copies/ml

1 copy /ml ≥ 1 copy/transfusion

400 ml

≥ 1 copy/transfusion

400 ml

NAT window period NAT window periodNAT positive

9.5 d 22.4 d 9.5 d15.2 d 13.9 d 5.8 d

Doubling time: 2.6 days Half-life: 1.6 days

Yoshizawa et al., modified

HBV NAT yield of HBsAg neg. donations from chronic carriers and window donors

12.8 Mill. 21 6 (22.2%)

Number of donations Pool NAT Missed by pool NAT tested positive

Window donations

15 13 2 (13%)

Chronic carriers

12 8 4 (33%)

VirusTM 7500

100 µlLC 480100 µl

TM 7500400 µl

LC 480400 µl

*Manufakt.850 µl

HCV 96 98 23 46 11

HIV-1 1184 868 41 27 49

HBV 64 53 5.4 4.4 3.8

HIV-2 375 cop./ml

92 cop./ml

n. d. n. d. n.a.

HAV 85 40 n. d. n. d. n.a.

B19 392 518 n. d. n. d. n.a.

100 µl / 850 µl input volume, direct extraction 400 µl input volume with centrifugation

Limit of Detection (95%) in IU/ml in individual donation testing

autoX-System ID Sensitivity

*Schmidt M, et al. Vox Sanguinis (2010) 98, 37-46; Pichl L, personal communication

GFE Blut Pool size

96(100 µl)*

24(400 µl)*

12(800 µl)*

HCV 1517 563 216

HIV-1 1770 79 59

HBV 129 16.5 5.5

Throughput(samples / run)

4320 1080 540

IU/ml individual donation plasma (centrifugation, LightCycler®480)

* Input volume per individual donation

Limit of Detection (95%) per individual donation in pools of different sizes

autoX-System Scalable Sensitivity

+Manufact.

SD(850 µl)*

11

49

3.8 (pools of 6 = 22.8)

+Schmidt M, et al. Vox Sanguinis (2010) 98, 37-46; Pichl L, personal communication94th BEPAC meeting April 2009

HIV-1 Transmissions in Germany

0

1

2

3

4

1998 1999 2000 2001 2002 2003 2004 2005 2006 2007

HIV-1 Transmissions HIV-1 NAT-only positive

HIV-1 NAT mandated

M. Nübling et al. Transfusion, 2009

No infection

• 2007 A commercial gag-region HIV-1 NAT test failed to detect a serologically positive donation. The previous donation by the same donor was serologically and NAT negative. The transfusion led to an infection in the recipient.

• 2009 A commercial LTR-region HIV-1 NAT test failed to detect a serologically negative donation. The GFE Blut VSPK v1.1 yielded a positive result for the donation in question and the serologically positive follow up donation. The transfusion led to an infection.

• 2010 The GFE Blut VSPK 1.1 failed to detect two serologically HIV-1 positive donations. An alternative clinical diagnostics PCR test yielded a positive result for these donations. No transmission occurred.

Afer sequencing, it could be determined that both cases were caused by the same multiple mutations. These mutations are not represented in the sequence databases.

Recent HIV-1 Test Failures

Wiesentheid GGAACCCACTGCTTAAGCCTCAATAAACTGTGCTTTGAGTGCTTCAAGTAGTGTGTGCCC Hagen2 GGAACCCACTGCTTAAGCCTCAATAAACTGTGCTTTGAGTGCTTCAAGTAGTGTGTGCCC Regensburg GGAACCCACTGCTTAAGCCTCAATAAACTGTGCTTTGAGTGCTTCAAGTAGTGTGTGCCC Hagen1 --------CTGCTTAAGCCTCAATAAAGCTTGCCTTGAGTGCTTAAAGTAGTGTGTGCCC HXB2 GGAACCCACTGCTTAAGCCTCAATAAAGCTTGCCTTGAGTGCTTCAAGTAGTGTGTGCCC ******************* *** ********** *************** Wiesentheid GTCTGTTGTGTGACTCTGGTAACTAGAGATCCCTCAGACACTTTTAGTCAGTGTGGAAAA Hagen2 GTCTGTTGTGTGACTCTGGTAACTAGAGATCCCTCAGACACTTTTAGTCAGTGTGGAAAA Regensburg GTCTGTTGTGTGACTCTGGTAACTAGAGATCCCTCAGACCCTTTTAGTCAGTGTGGAAAA Hagen1 GTCTGTTGTGTGACTCTGGTAACTAGAGATCCCTCAGACCTTTATAGTCAGTGTGGAAAA HXB2 GTCTGTTGTGTGACTCTGGTAACTAGAGATCCCTCAGACCCTTTTAGTCAGTGTGGAAAA *************************************** ** **************** Wiesentheid TCTCTAGCAGTGGCGCCCGAACAGGGACCTGAAAACGAAAGTAAGACCAGAGAAGATCTC Hagen2 TCTCTAGCAGTGGCGCCCGAACAGGGACCTGAAAACGAAAGTAKGACCASAGAAGTTCTC Regensburg TCTCTAGCAGTGGCGCCCGAACAGGGACTCGAAAGCGAAAGTAAGACCAGAGAAGTTCTC Hagen1 TCTCTAGCAGTGGCGCCCGAACAGGGACTTGAAAGTGAAAATAAGACCAGAGGAGCTCTC HXB2 TCTCTAGCAGTGGCGCCCGAACAGGGACCTGAAAGCGAAAGGGAAACCAGAGGAGCTCTC **************************** **** **** **** ** ** ****

Sequence comparison of HIV-1 isolates:

2010_12010_2Reg.2009Consensus

2010_12010_2Reg.2009Consensus

2010_12010_2Reg.2009Consensus

HIV-1 Sequence Alignment

HIV-1 PCR v1Reference (+)Isolate 1 (-)Isolate 2 (-)

Sensitivity of different HIV-1 LTR PCR versions

HIV-1 PCR v2Reference (+)Isolate 1 (+)Isolate 2 (+)

Reference (-)Isolate 1 (+)Isolate 2 (+)

HIV-1 PCR (new v2 primer only)

R

2

R

1

2

1

R

1/2

HIV-1 PCR Assay Improvements

env sequences of the new isolates form a unique cluster of HIV-1 B subtypes

2

HIV-1 Philogenetic Analysis

pr rt sequences of the new isolates confirm a separate cluster of subgroup B strains together with the related clinical strain

HIV-1 Philogenetic Analysis

2010 -1

2010 -2

2010 clin.

Sensitivity is sufficient and can easily be adapted to new requirements

New virus variants evolve rapidly

Newly evolving variants pose a signifcant risk to subtype coverage by NAT tests

Great care must be taken, not to create more problems with the a new PCR version than are solved.

Due to the quick evolution of the target sequences, vigilance remains a prime directive.

As has been proven, it is possible to release an updated kit version very quickly. But apart from a lot of hard work, it takes the cooperation of all parties involved to achieve this.

Summary „Old“ Portfolio Viruses

We Thank

• DRK BSD West– Dr. Lutz Pichl– Dr. Volkmar Schottstedt– Dr. Peter Muss– Petra Niggemeier– Prof. Dr. Jürgen Bux

• BRK BSD Bayern, Institut Wiesentheid– Dr. Marijke Weber-Schehl– Dr. Doris Hedges– Dr. Franz Weinauer

• Ludwig-Maximilians-University– Prof. Dr. Josef Eberle

• Paul-Ehrlich-Institut– Dr. Micha Nübling– Dr. Margarethe Heiden– Dr. Markus Funk

• mdc– Dr. Michael Dreher

Challenges for NAT Testing

• Sensitivity• Throughput• Flexibility• Viruses

– „Old“ portfolio viruses• HCV• HBV• HIV-1

– „New“ portfolio viruses• Human Cytomegalo Virus• West Nile Virus• Chikungunya Virus• Dengue

Human Cytomegalovirus (HCMV)

• Human cytomegalovirus (HCMV) is

ubiquitous around the world!

• The seroprevalence in the population increases with age, with 10 - 20 % of children infected before they reach puberty, while by adulthood, the prevalence of HCMV is 40 – 100 %.

• Causes mostly asymptomatic or mild mononucleosis-like infections in immune competent patients.

• Causes severe infections in immuno-compromised patients and during pregnancy

• High risk of transmission upon blood transfusion

IMMUNOCOMPROMISED PATIENTSNearly all HIV-infected patients are infected with CMV

PREGNANT WOMEN

ORGAN TRANSPLANT PATIENTS

IMMUNOCOMPETENT PATIENTSMostly asymptomatic

CMV+

X

X

X

?Child of women with primary CMV infection during pregnancy

Significant cause of morbidity in bone marrow transplant and solid organ transplant patients

HCMV: Blood Recipients at Risk

MEASURES to REDUCE HCMV TRANSMISSION RISKS:

• Leucodepletion

• Antibody testing

• NAT testing

• High flexibility through inclusion into the autoX- System: variable pools up to 96 individual donations; plasma input from 100 µl (minimum) up to 400 µl (maximal sensitivity)

• Multiplex compatibility with other viruses in the GFE Blut Virus Screening PCR Kit (VSPK), i.e. Pb19, HBV, HCV

• High specificity

• High sensitivity, comparable to other commercially available HCMV-PCRs.

• Applicable as manual as well as automated system (autoX- System)

• In development: available at the end of 2011

HCMV-PCR Kit

Depending on regional epidemiology, screening for other viruses is important. Due to changes in climate, combined with constantly increasing inter-continental travelling activities, the need may arise to test for (sub-)tropical viruses in Europe, too.

For example, cases of Chikungunya infections have been reported from Northern Italy. The mosquito vector has already invaded Europe.

A research cooperation between the Bernhard-Nocht-Institut für Tropenmedizin and the Senckenberg Deutsches Entomologisches Institut (SDEI) has been launched to determine the distribution of West Nile Virus in German mosquito populations.

Therefore, GFE Blut is near the completion of kits for the detection of ChikV, WNV and Dengue, in order to be able to reduce the reaction time, if the need to screen for these viruses turns into an urgent issue.

„New“ Portfolio Viruses

The current GFE Blut autoX-System is designed for the specifc needs of the German blood donation environment, with a high degree of centralisation and hence for high-throughput testing.

New challenges arise from “Old” portolio viruses to cope with: Increased analytical and clinical sensitivity, new genetic variants.

New challenges arise from “New” portolio viruses due to climate change, global trading and travelling

Other regions, with a different epidemiology, different geographical / infra-structural conditions, would need platorms optimised for their specifc needs, which ofen means optimised for lower through-put and higher sensitivity without compromising safety or cost-efficiency.

To complement our portolio, GFE Blut will be developing new kits and platorms which will be better suited for these circumstances.

Summary