Original Article Transfus Med Hemother 2014;41:45–51 DOI: 10.1159/000357103
Received: July 02, 2013 Accepted: October 16, 2013 Published online: Dezember 19, 2013
Risk Minimization Measures for Blood Screening HIV-1 Nucleic Acid Amplification Technique Assays in Germany Michael Chudya Julia Kressa Jochen Halbauerb Margarethe Heidenc Markus B. Funkb C. Micha Nüblinga a
Section of Molecular Virology, Section of Hemovigilance and IVD Vigilance, c Section of Transfusion Medicine, Paul-Ehrlich-Institut, Langen, Germany b
Keywords HIV-1 nucleic acid amplification technique · Blood safety · HIV-1 variant Summary Background: Several publications describe HIV-1 RNA false-negative results or viral load underquantitation associated with Communauté Européenne(CE)-marked qualitative or quantitative nucleic acid amplification technique (NAT) assays. 6 cases occurred during blood screening in Germany, with 2 of them causing HIV-1 transmissions to recipients of blood components. The implicated NAT assays were mono-target assays amplifying in different viral genome regions (gag or long terminal repeat). Methods: Specimens characterized by HIV-1 NAT underquantitation or false-negative NAT results were comparatively investigated in CE-marked HIV-1 NAT systems of different design to identify potential reasons. The target regions of the viral nucleic acids were sequenced and these sequences compared to primers and probes of the assays. Potential risk minimization measures were considered for quantitative and blood-screening HIV-1 NAT systems. Results: Nucleotide sequencing of the viral target region in cases of HIV-1 RNA underquantitation or false-negative test results revealed new HIV-1 variants that were mismatched with primers and probes used in some mono-target assays. So far, dualtarget NAT assays have not been associated with mismatch-based false-negative test results. From 2015, the Paul Ehrlich Institute will request HIV-1 NAT assays of dual-target design or an analogous solution for further reducing the risk in blood screening. Conclusion: HIV differs from other blood-borne viruses with regard to its fast
© 2014 S. Karger GmbH, Freiburg 1660-3796/14/0411-0045$39.50/0 Fax +49 761 4 52 07 14 [email protected] www.karger.com
Accessible online at: www.karger.com/tmh
evolution of new viral variants. The evolution of new sequences is hardly predictable; therefore, NAT assays with only 1 target region appear to be more vulnerable to sequence variations than dual-target assays. The associated risk may be higher for HIV-1 NAT assays used for blood screening compared to quantitative assays used for monitoring HIV-1-infected patients. In HIV-1 screening NAT assays of dual-target design may adequately address the risk imposed by new HIV-1 variants.
Introduction As summarized by the European Center for Disease Control (ECDC) in its most recent report [1], HIV infection remains one of the major public health problems in Europe. Nevertheless, on a global scale, the overall HIV epidemiology in Europe appears moderate. The HIV epidemiology in risk groups differs between countries. At present, in Europe, men who have sex with men (MSM) comprise the largest group of cases (38%), followed by those who acquired the virus through heterosexual contact (24%) and injecting drug users (4%). Cases in MSM increased by 39% between 2004 and 2010, while cases acquired by heterosexual transmission or in other risk groups have remained stable or are declining. Based on the introduction of highly sensitive test systems for donor screening, the risk for infection via blood transfusion has become very low in Europe. In Germany, both the general and the blood donor population are characterized by low prevalence and incidence rates when compared to the global HIV situation. For blood donors
Dr. C. Micha Nübling Section of Molecular Virology Paul-Ehrlich-Institut Paul-Ehrlich-Straße 51–59, 63225 Langen, Germany [email protected]
Table 1. CE-marked HIV-1 NAT assaysa Abbreviation
Name
Type
Manufacturer
CAP CTM v1 HPS CTM v1 CAS v1.5 CAM v1.5 CTS MPX VSPK v1.1 artus Abbott RT VSPK v1.2 CAP CTM v2 Ultrio Plus
COBAS AmpliPrep/COBAS TaqMan HIV-1 test HPS Viral Nucleic Acid Kit/COBAS TaqMan HIV-1 test COBAS AmpliScreen HIV-1 test v1.5 COBAS Amplicor HIV-1 monitor test, v1.5 cobas TaqScreen MPX test for use with cobas S201 system Virus screening PCR kit v1.1 artus HIV-1: RG RT-PCR kit Abbott real time HIV-1 assay Virus screening PCR kit v1.2 COBAS AmpliPrep/COBAS TaqMan HIV-1 test, v2.0 Procleix Ultrio Plus assay
quantitative quantitative qualitative quantitative qualitative qualitative quantitative quantitative qualitative quantitative qualitative
Roche Molecular Systems, Pleasanton, CA Roche Molecular Systems, Pleasanton, CA Roche Molecular Systems, Pleasanton, CA Roche Molecular Systems, Pleasanton, CA Roche Molecular Systems, Pleasanton, CA GFE Blut, Frankfurt/M. Qiagen GmbH, Hilden Abbott Molecular, Des Plaines, IL GFE Blut, Frankfurt/M. Roche Molecular Systems, Pleasanton, CA Gen-Probe, San Diego, CA
CE = Communauté Européenne, NAT = nucleic acid amplification technique. The CAM v1.5 assay is regarded as representative for the related assays (Amplicor HIV-1 Monitor Test, v1.5 and COBAS AmpliPrep/ COBAS Amplicor HIV-1 Monitor Test, v1.5) because of the common amplification module; the Ultrio Plus assay is regarded as representative for the related Procleix Ultrio Assay, which has the same HIV-1 amplification features.
a
in Germany the most recent published data describe, for the period 2008–2010, an HIV prevalence in first-time donors (FTD) of 6.8 per 100,000 applicant donors, and an HIV seroconversion rate of 2.4 per 100,000 repeat donors [2]. Continuous efforts to prevent transfusion-associated HIV-1 transmissions led to the introduction of NAT tests for blood screening in addition to serological assays. Although some blood donation centers had already introduced HIV-1 NAT in the mid90s on a voluntary basis [3], the Paul Ehrlich Institute (PEI) required HIV-1 NAT from 2004 on, with a minimal sensitivity limit for the individual donation of 10,000 IU HIV-1 RNA/ml (based on the WHO International Standard for HIV-1 RNA) [4, 5]. In Germany, this NAT requirement facilitates pooling of specimens prior to NAT testing and the use of different validated assay types for blood screening including Communauté Européenne(CE)-marked diagnostic assays of high sensitivity or in-house-developed screening assays [6]. After some years of NAT testing experience in Germany, the NAT yield (donations from the diagnostic window phase: NAT positive, anti-HIV negative) was determined [7] and recently updated. From 2004 to December 2010, 23 HIV-1 NAT yield cases were found in more than 31 million NAT-screened donations, but 2 HIV-1 transmissions, despite NAT testing, were observed during this period. These HIV-1 transmissions should have been interdicted by NAT due to the high viral loads present in the donors. The cases were traceable to falsenegative test results in the routine NAT assays due to new variants of the common HIV-1 subtype B missed by the assays’ design. Recently, details of these 2 transmission cases and a number of related cases of false-negative HIV-1 NAT screening results have been published [8–12]. In addition, we must emphasize that cases of false-negative HIV-1 NAT based on low viral load combined with suboptimal NAT sensitivity in the screening of mini pools cannot be excluded. Fortunately, until now, respective transmissions have not been
46
Transfus Med Hemother 2014;41:45–51
experienced in Germany. However, a residual risk of HIV transmission exists even with NAT performed for each individual donation (ID-NAT). There have been several reports of underquantitation of the viral load by CE-marked quantitative (diagnostic) HIV-1 NAT, traceable also to the assays’ design [13–16]. These false-negative or underquantified test results occurred with screening or diagnostic NAT assays targeting 1 region of the HIV-1 genome in combination with new HIV-1 variants. The nucleic acid sequences of these new HIV-1 subtype B variants were not available at the time of assay design. In some cases, in vitro diagnostic medical device (IVD) manufacturers reacted promptly by adapting the assay design, either by adapting primer/probe sequences for inclusion of new viral variants [9] or by inclusion of a second target region into the assay design [17, 18]. Potential risk minimization measures have been discussed at both the European and national regulatory level. While risk assessment for quantitative diagnostic HIV-1 NAT assays negated the necessity of immediate regulatory consequences at the European level, risk assessment for blood-screening HIV-1 NAT assays resulted in our requirement for dual-target design, or analogous safety solution, at national level. This requirement has been defined in the context of quality control of blood components, which are regulated under the German Drug Law. A similar discussion is currently underway at the European level regarding a potential update of the Common Technical Specifications for qualitative HIV-1 NATs [19].
Material and Methods Plasma Specimens Blood collection centers provided samples from blood donations that tested falsely negative in CE-marked NAT assays used for screening (cases 1–6). The viral load was determined using different quantitative HIV-1 NATs targeting different regions of the viral genome. The mean
Chudy/Kress/Halbauer/Heiden/Funk/Nübling
Fig. 1. Alignment of +;% &7***$*&7&7&7**&7$$&7$***$$&&&$&7*&77$$*&&7&$$7$$$*&77*&&77* HIV-1 long terminal 3(,5HI &DVH repeat (LTR) se&DVH &7*7 quences. Compari&DVH &7*7 son of partial 5’ LTR &DVH * sequences (nucle otides 483–725) de+;% $*7*&77&$$*7$*7*7*7*&&&*7&7*77*7*7*$&7&7**7$$&7$*$*$7&&&7&$* rived from amplified 3(,5HI * genomes of the Paul&DVH $ Ehrlich Institute &DVH (PEI) HIV-1 RNA &DVH reference prepara&DVH $$&7& tion (#3441/04; sub type B) and samples +;% $&&&7777$*7&$*7*7**$$$$7&7&7$*&$*7**&*&&&*$$&$***$&&7*$$$*&* 3(,5HI 7 from non-detection &DVH 7$77 cases 3–6 with the se&DVH $$ quence of the HIV-1 &DVH $$ prototype HXB2 &DVH 777 (accession number K03455). Dashes +;% $$$***$$$&&$*$**$*&7&7&7&*$&*&$**$&7&**&77*&7*$$*&*&*&$&**&$$*$ represent homology. 3(,5HI 7*$ Differences are &DVH $7$*&$* shown by the appro&DVH 7$*$$
Received: July 02, 2013 Accepted: October 16, 2013 Published online: Dezember 19, 2013
Risk Minimization Measures for Blood Screening HIV-1 Nucleic Acid Amplification Technique Assays in Germany Michael Chudya Julia Kressa Jochen Halbauerb Margarethe Heidenc Markus B. Funkb C. Micha Nüblinga a
Section of Molecular Virology, Section of Hemovigilance and IVD Vigilance, c Section of Transfusion Medicine, Paul-Ehrlich-Institut, Langen, Germany b
Keywords HIV-1 nucleic acid amplification technique · Blood safety · HIV-1 variant Summary Background: Several publications describe HIV-1 RNA false-negative results or viral load underquantitation associated with Communauté Européenne(CE)-marked qualitative or quantitative nucleic acid amplification technique (NAT) assays. 6 cases occurred during blood screening in Germany, with 2 of them causing HIV-1 transmissions to recipients of blood components. The implicated NAT assays were mono-target assays amplifying in different viral genome regions (gag or long terminal repeat). Methods: Specimens characterized by HIV-1 NAT underquantitation or false-negative NAT results were comparatively investigated in CE-marked HIV-1 NAT systems of different design to identify potential reasons. The target regions of the viral nucleic acids were sequenced and these sequences compared to primers and probes of the assays. Potential risk minimization measures were considered for quantitative and blood-screening HIV-1 NAT systems. Results: Nucleotide sequencing of the viral target region in cases of HIV-1 RNA underquantitation or false-negative test results revealed new HIV-1 variants that were mismatched with primers and probes used in some mono-target assays. So far, dualtarget NAT assays have not been associated with mismatch-based false-negative test results. From 2015, the Paul Ehrlich Institute will request HIV-1 NAT assays of dual-target design or an analogous solution for further reducing the risk in blood screening. Conclusion: HIV differs from other blood-borne viruses with regard to its fast
© 2014 S. Karger GmbH, Freiburg 1660-3796/14/0411-0045$39.50/0 Fax +49 761 4 52 07 14 [email protected] www.karger.com
Accessible online at: www.karger.com/tmh
evolution of new viral variants. The evolution of new sequences is hardly predictable; therefore, NAT assays with only 1 target region appear to be more vulnerable to sequence variations than dual-target assays. The associated risk may be higher for HIV-1 NAT assays used for blood screening compared to quantitative assays used for monitoring HIV-1-infected patients. In HIV-1 screening NAT assays of dual-target design may adequately address the risk imposed by new HIV-1 variants.
Introduction As summarized by the European Center for Disease Control (ECDC) in its most recent report [1], HIV infection remains one of the major public health problems in Europe. Nevertheless, on a global scale, the overall HIV epidemiology in Europe appears moderate. The HIV epidemiology in risk groups differs between countries. At present, in Europe, men who have sex with men (MSM) comprise the largest group of cases (38%), followed by those who acquired the virus through heterosexual contact (24%) and injecting drug users (4%). Cases in MSM increased by 39% between 2004 and 2010, while cases acquired by heterosexual transmission or in other risk groups have remained stable or are declining. Based on the introduction of highly sensitive test systems for donor screening, the risk for infection via blood transfusion has become very low in Europe. In Germany, both the general and the blood donor population are characterized by low prevalence and incidence rates when compared to the global HIV situation. For blood donors
Dr. C. Micha Nübling Section of Molecular Virology Paul-Ehrlich-Institut Paul-Ehrlich-Straße 51–59, 63225 Langen, Germany [email protected]
Table 1. CE-marked HIV-1 NAT assaysa Abbreviation
Name
Type
Manufacturer
CAP CTM v1 HPS CTM v1 CAS v1.5 CAM v1.5 CTS MPX VSPK v1.1 artus Abbott RT VSPK v1.2 CAP CTM v2 Ultrio Plus
COBAS AmpliPrep/COBAS TaqMan HIV-1 test HPS Viral Nucleic Acid Kit/COBAS TaqMan HIV-1 test COBAS AmpliScreen HIV-1 test v1.5 COBAS Amplicor HIV-1 monitor test, v1.5 cobas TaqScreen MPX test for use with cobas S201 system Virus screening PCR kit v1.1 artus HIV-1: RG RT-PCR kit Abbott real time HIV-1 assay Virus screening PCR kit v1.2 COBAS AmpliPrep/COBAS TaqMan HIV-1 test, v2.0 Procleix Ultrio Plus assay
quantitative quantitative qualitative quantitative qualitative qualitative quantitative quantitative qualitative quantitative qualitative
Roche Molecular Systems, Pleasanton, CA Roche Molecular Systems, Pleasanton, CA Roche Molecular Systems, Pleasanton, CA Roche Molecular Systems, Pleasanton, CA Roche Molecular Systems, Pleasanton, CA GFE Blut, Frankfurt/M. Qiagen GmbH, Hilden Abbott Molecular, Des Plaines, IL GFE Blut, Frankfurt/M. Roche Molecular Systems, Pleasanton, CA Gen-Probe, San Diego, CA
CE = Communauté Européenne, NAT = nucleic acid amplification technique. The CAM v1.5 assay is regarded as representative for the related assays (Amplicor HIV-1 Monitor Test, v1.5 and COBAS AmpliPrep/ COBAS Amplicor HIV-1 Monitor Test, v1.5) because of the common amplification module; the Ultrio Plus assay is regarded as representative for the related Procleix Ultrio Assay, which has the same HIV-1 amplification features.
a
in Germany the most recent published data describe, for the period 2008–2010, an HIV prevalence in first-time donors (FTD) of 6.8 per 100,000 applicant donors, and an HIV seroconversion rate of 2.4 per 100,000 repeat donors [2]. Continuous efforts to prevent transfusion-associated HIV-1 transmissions led to the introduction of NAT tests for blood screening in addition to serological assays. Although some blood donation centers had already introduced HIV-1 NAT in the mid90s on a voluntary basis [3], the Paul Ehrlich Institute (PEI) required HIV-1 NAT from 2004 on, with a minimal sensitivity limit for the individual donation of 10,000 IU HIV-1 RNA/ml (based on the WHO International Standard for HIV-1 RNA) [4, 5]. In Germany, this NAT requirement facilitates pooling of specimens prior to NAT testing and the use of different validated assay types for blood screening including Communauté Européenne(CE)-marked diagnostic assays of high sensitivity or in-house-developed screening assays [6]. After some years of NAT testing experience in Germany, the NAT yield (donations from the diagnostic window phase: NAT positive, anti-HIV negative) was determined [7] and recently updated. From 2004 to December 2010, 23 HIV-1 NAT yield cases were found in more than 31 million NAT-screened donations, but 2 HIV-1 transmissions, despite NAT testing, were observed during this period. These HIV-1 transmissions should have been interdicted by NAT due to the high viral loads present in the donors. The cases were traceable to falsenegative test results in the routine NAT assays due to new variants of the common HIV-1 subtype B missed by the assays’ design. Recently, details of these 2 transmission cases and a number of related cases of false-negative HIV-1 NAT screening results have been published [8–12]. In addition, we must emphasize that cases of false-negative HIV-1 NAT based on low viral load combined with suboptimal NAT sensitivity in the screening of mini pools cannot be excluded. Fortunately, until now, respective transmissions have not been
46
Transfus Med Hemother 2014;41:45–51
experienced in Germany. However, a residual risk of HIV transmission exists even with NAT performed for each individual donation (ID-NAT). There have been several reports of underquantitation of the viral load by CE-marked quantitative (diagnostic) HIV-1 NAT, traceable also to the assays’ design [13–16]. These false-negative or underquantified test results occurred with screening or diagnostic NAT assays targeting 1 region of the HIV-1 genome in combination with new HIV-1 variants. The nucleic acid sequences of these new HIV-1 subtype B variants were not available at the time of assay design. In some cases, in vitro diagnostic medical device (IVD) manufacturers reacted promptly by adapting the assay design, either by adapting primer/probe sequences for inclusion of new viral variants [9] or by inclusion of a second target region into the assay design [17, 18]. Potential risk minimization measures have been discussed at both the European and national regulatory level. While risk assessment for quantitative diagnostic HIV-1 NAT assays negated the necessity of immediate regulatory consequences at the European level, risk assessment for blood-screening HIV-1 NAT assays resulted in our requirement for dual-target design, or analogous safety solution, at national level. This requirement has been defined in the context of quality control of blood components, which are regulated under the German Drug Law. A similar discussion is currently underway at the European level regarding a potential update of the Common Technical Specifications for qualitative HIV-1 NATs [19].
Material and Methods Plasma Specimens Blood collection centers provided samples from blood donations that tested falsely negative in CE-marked NAT assays used for screening (cases 1–6). The viral load was determined using different quantitative HIV-1 NATs targeting different regions of the viral genome. The mean
Chudy/Kress/Halbauer/Heiden/Funk/Nübling
Fig. 1. Alignment of +;% &7***$*&7&7&7**&7$$&7$***$$&&&$&7*&77$$*&&7&$$7$$$*&77*&&77* HIV-1 long terminal 3(,5HI &DVH repeat (LTR) se&DVH &7*7 quences. Compari&DVH &7*7 son of partial 5’ LTR &DVH * sequences (nucle otides 483–725) de+;% $*7*&77&$$*7$*7*7*7*&&&*7&7*77*7*7*$&7&7**7$$&7$*$*$7&&&7&$* rived from amplified 3(,5HI * genomes of the Paul&DVH $ Ehrlich Institute &DVH (PEI) HIV-1 RNA &DVH reference prepara&DVH $$&7& tion (#3441/04; sub type B) and samples +;% $&&&7777$*7&$*7*7**$$$$7&7&7$*&$*7**&*&&&*$$&$***$&&7*$$$*&* 3(,5HI 7 from non-detection &DVH 7$77 cases 3–6 with the se&DVH $$ quence of the HIV-1 &DVH $$ prototype HXB2 &DVH 777 (accession number K03455). Dashes +;% $$$***$$$&&$*$**$*&7&7&7&*$&*&$**$&7&**&77*&7*$$*&*&*&$&**&$$*$ represent homology. 3(,5HI 7*$ Differences are &DVH $7$*&$* shown by the appro&DVH 7$*$$
