Accepted Manuscript Title: Mini review:Current molecular methods for the detection and quantification of hepatitis B virus, hepatitis C virus, and human immunodeficiency virus type 1 Author: Guilherme Albertoni Manoel Jo˜ao Batista Castelo Gir˜ao Nestor Schor PII: DOI: Reference:
S1201-9712(14)01502-1 http://dx.doi.org/doi:10.1016/j.ijid.2014.04.007 IJID 1979
To appear in:
International Journal of Infectious Diseases
Received date: Revised date: Accepted date:
6-3-2014 1-4-2014 9-4-2014
Please cite this article as: Guilherme AlbertoniManoel Jo˜ao Batista Castelo Gir˜aoNestor Schor Mini review:Current molecular methods for the detection and quantification of hepatitis B virus, hepatitis C virus, and human immunodeficiency virus type 1 (2014), http://dx.doi.org/10.1016/j.ijid.2014.04.007 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Running heads: Recto: Molecular methods for detection of viruses
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Verso: G. Albertoni et al.
Correspondence footnote:
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[ARTICLE TYPE – REVIEW ARTICLE]
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E-mail address: [email protected] (G. Albertoni).
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*Corresponding author. Tel.: +55 11 993647519; fax: +55 11 50556588.
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Mini review: Current molecular methods for the detection and quantification of hepatitis B virus, hepatitis C virus, and
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human immunodeficiency virus type 1
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Guilherme Albertonia,b,*, Manoel João Batista Castelo Girãob, Nestor Schora
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Federal University of São Paulo (UNIFESP), Department of Medicine, Nephrology
Division, Rua Botucatu 740, São Paulo, 04023-900, SP, Brazil b
Colsan (Associação Beneficente de Coleta de Sangue), São Paulo, SP, Brazil
[Received date] Corresponding Editor: Larry Lutwick, Kalamazoo, Michigan, USA
KEYWORDS
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NAT testing; HIV; HCV;
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HBV
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Summary
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The detection of acute human immunodeficiency virus (HIV), hepatitis B virus
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(HBV), and hepatitis C virus (HCV) infection is vital for controlling the spread of HIV, HBV, and HCV to uninfected individuals. Considering that these viruses have
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high replication rates and are undetectable by serological markers, early detection upon transmission is crucial. Various nucleic acid assays have been developed for
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diagnostics and therapeutic monitoring of infections. In the past decade, rapid and
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sensitive molecular techniques such as PCR have revolutionized the detection of a variety of infectious viruses, including HIV, HCV, and HBV. Here, we describe two
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of the most commonly used licensed methods for the detection and quantification of HIV, HCV, and HBV: the cobas TaqScreen MPX (PCR) test and the Tigris System. We used transcription-mediated amplification to review and compare the development and efficiency of these technologies.
Introduction
The quality and safety of blood products used in transfusions are major public health concerns. In addition to general quality control (QC), the introduction of good manufacturing practices and routine screening of blood materials and products, has
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ensured consistency, quality, and safety in the increased production and use of blood products in recent decades. Newly developed serological tests and nucleic acid tests (NATs) have markedly reduced the risk of transmission of human immunodeficiency
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virus (HIV), hepatitis C virus (HCV), and hepatitis B virus (HBV).1 In the past decade, rapid and sensitive molecular techniques such as PCR have revolutionized the
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detection of a variety of infectious viruses.2–4
About 10 years ago, molecular methods achieved a significant level of
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throughput and automation, enabling their introduction as an additional tool in blood screening. The aim of these tests was primarily to prevent donations made during the
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window-period from being used in transfusions.5,6 An international survey7 reported
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findings from the use of NATs in 330 million blood donations and showed overall infection rates of 1:447 000 for HCV, 1:111 000 for HIV, and 1:66 000 for HBV. Up
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until 2011, very few of Brazil’s blood banks (corresponding to less than 5% of the
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national blood supply) had voluntarily introduced NAT screening. More recently, an HCV/HIV NAT kit was developed by Bio-Manguinhos/Fiocruz (Rio de Janeiro,
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Brazil). This test is now used in some of Brazil’s large public blood banks and is expected to be extended to all public blood banks by 2012 when the test may become mandatory [Au?1].8
In this article, we describe two of the most commonly used licensed molecular
methods for the detection and quantification of HIV, HCV, and HBV in blood. We compared the lower limits of virus detection of each method and evaluated the overall performance.
Nucleic acid testing (NAT)
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The World Health Organization (WHO) recommends that diagnostic devices are ‘ASSURED’: Affordable, Sensitive, Specific, User-friendly, Rapid and Robust, Equipment-free, and Deliverable to end users, especially with regard to developing
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countries. Nucleic acid amplification techniques (NAATs), however, typically require a significant investment in equipment, training, and infrastructure. Nevertheless, the
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development of these molecular techniques as diagnostic tools has become
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increasingly important.9–13
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Real-time PCR
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The fact that PCR reactions can be monitored in real time has revolutionized the process of DNA and RNA fragment quantification. Nucleic acid quantification using
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quantitative real-time PCR (qRT-PCR) is extremely accurate and reproducible. In
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qRT-PCR, each round of amplification leads to the emission of a fluorescent signal, and the number of signals per cycle is proportional to the amount of target nucleic
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acid in the starting sample.16 [Au?2] This allows for accurate and reproducible quantification based on fluorescence. The most commonly used fluorescent compounds in qRT-PCR are the SYBR Green dye and the TaqMan probe.16 qRT-PCR
requires an instrumentation platform that consists of a thermal cycler, a computer, optics for fluorescence excitation and emission collection, and data acquisition and analysis software.14–16
In-house NAT for HCV detection
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For the detection and quantification of HCV RNA,17 we designed and developed a one-step NAT TaqMan qRT-PCR method. Using international standards (ACCURUN HCV RNA Positive Control;
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SeraCare) containing 3 × 105 IU/ml of HCV RNA (1 U = 1–3 copies),14 our NAT inhouse qRT-PCR TaqMan HCV assay displayed linearity over a range of 3.1 × 102 to
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3.1 × 105 IU/ml (2.5–5.5 log). The lower limit of detection of the method was 310
IU/ml. In a previous report by Levi et al.,18 [Au?3] the method used to screen blood
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donors by NAT testing for HCV RNA displayed linearity over a range of 5 × 102 to 5 × 104. The lower limit of detection of the method was 500 IU/ml. In another study,
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Paryan et al.19 designed and developed an in-house multiplex RT-PCR assay for the
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detection of HCV in plasma samples. That multiplex assay was linear between 102 and 105 copies/ml (six of eight samples were detectable), and had an analytical
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sensitivity of 200 copies/ml.
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In-house NAT for HIV detection
We designed and developed a one-step TaqMan NAT qRT-PCR method for the detection of HIV RNA.20
We recently developed a NAT TaqMan qRT-PCR HIV RNA assay using a set
of international standards (AcroMetrix HIV-1 Panel IU/ml RNA Positive Control; Applied Biosystems) containing 1 × 102 to 1 × 107 IU/ml of HIV RNA. Each panel member (except the negative control) contains HIV-1 at a predetermined concentration calibrated against the WHO international standard for HIV-1 RNA. Consequently, the assigned values are reported in international units per milliliter (IU/ml). Some studies have reported results in units that differ from the international
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units as defined by the WHO.21 For our in-house NAT TaqMan qRT-PCR HIV assay, we observed linearity over a range of 1 × 102 to 1 × 107 IU/ml and a lower limit of detection of 100 IU/ml (Figure 1). Similarly, Paryan et al.,19 using multiplex RT-PCR,
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showed linearity over a range of 1 × 102 to 1 × 105 copies/ml and an analytical sensitivity of 100 copies/ml for HIV RNA. In experiments by De Cringes et al.,22
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[Au?4] a SYBR Green I Multiplex RT-PCR was successfully used to detect HIV
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RNA, and was found to have an analytical sensitivity of 400 copies/ml for HIV RNA.
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[Figure 1 here]
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In-house NAT for HBV detection
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We developed a NAT in-house qRT-PCR TaqMan HBV DNA assay using
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international standards (AcroMetrix HBV Panel IU/ml DNA Positive Control; Applied Biosystems) containing 2 × 102 to 2 × 107 IU/ml of HBV DNA. Our HBV
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assay displayed linearity over a range of 2 × 102 to 2 × 107 IU/ml, and the lower limit of detection of the method was 200 IU/ml (Figure 2 [Au?5]). dos Santos et al.23 developed a cost-effective RT-PCR test to detect a wide range of HBV DNA in the western Amazonas region. Their method displayed linearity over a range of 2 × 103 to 2 × 109 copies/ml and a lower limit of detection of 2000 IU/ml. Yalamanchili et al.24
designed a rapid, reliable, and sensitive assay for the detection of HBV by RT-PCR. The calibration curve was linear over a range of 1 × 102 to 1 × 108 copies/ml, with an
R2 value of 0.999. The lower limit of detection of the method was 100 copies/ml.
[Figure 2 here]
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Current approaches
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Several US Food and Drug Administration (FDA)-licensed NAT assays are currently available for the screening of blood donors for HIV, HCV, HBV, and West Nile virus
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(WNV). The continued development of highly sensitive screening NAT systems,
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however, is challenging.25 The FDA requires that blood screening has licensed several triplex (HIV/HCV/HBV) automated NAT systems for blood donor screening
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[Au?6].26 These include the PCR-based cobas TaqScreen MPX assay using the cobas s 201 instrument (Roche Diagnostics GmbH, Mannheim, Germany), and the Procleix
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Ultrio assay, using the Procleix Tigris automated instrument (Novartis Diagnostics, Emeryville, CA, USA/Gen-Probe, San Diego, CA, USA) and employing
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transcription-mediated amplification (TMA).27–30
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PCR-based cobas TaqScreen MPX test
The cobas TaqScreen MPX test is a multiplex, multi-dye nucleic acid amplification technology. Sample nucleic acids and armored RNA internal control (IC) molecules, which serve as specimen preparation and amplification/detection process controls, are processed simultaneously. A protease solution digests proteins to promote lysis, inactivate nucleases, and facilitate the release of RNA and DNA from viral particles. The released nucleic acids bind to the silica surface of magnetic glass particles, which are added to the reaction mixture. The binding of nucleic acids to the particles is mainly due to the net positive charge on the glass particle surface and net negative charge of the nucleic acids at the chaotropic salt concentration and ionic strength of
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the lysis reaction. A wash reagent removes unbound substances and impurities. Using an elution buffer, purified nucleic acids are then released from the magnetic glass particles at high temperatures.31
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After isolation of the purified nucleic acids from human plasma, cobas TaqScreen Master Mix (MPX MMX) is used for the amplification and detection of
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HIV-1 (groups M and O), HIV-2, and HCV RNA, as well as HBV DNA and IC RNA. Once the MPX MMX is activated by the addition of manganese acetate, reverse
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transcription (for RNA targets) proceeds, followed by PCR amplification of highly conserved regions of HIV-1 (groups M and O), HIV-2, and HCV RNA, as well as
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HBV DNA and IC RNA using specific primers.31
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Simultaneous detection of the amplified nucleic acids is accomplished by the generation of fluorescent signals from 5′-nucleolytic degradation of HIV-1- (groups
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M and O), HIV-2-, HCV-, HBV- and IC-specific probes. Two fluorescent dyes are
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used, one labeling the IC probe and a second labeling all target-specific probes, allowing for the combined identification of each viral target as well as independent
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identification of the IC. Reverse transcription and amplification reactions are performed with a thermostable recombinant DNA polymerase enzyme. In the presence of manganese (Mn2+), DNA polymerase has reverse transcriptase and DNA polymerase activities. This allows both reverse transcription and PCR amplification to occur in the same reaction mixture.31 Selective amplification of target nucleic acid from the specimen is achieved by
the use of a uracil-N-glycosylase enzyme and deoxyuridine triphosphate. The enzyme recognizes and catalyzes the destruction of DNA strands containing deoxyuridine,32 but not DNA containing deoxythymidine or RNA containing ribouridine.33,34
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During PCR amplification, the intermittent high temperatures during thermal cycling denature target and IC amplicons to form single-stranded DNA. The specific oligonucleotide probes, which facilitate signal detection, hybridize to the single-
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stranded form of the amplified DNA, and amplification, hybridization, and detection occur simultaneously.33,35,36 During PCR amplification, probes hybridize to specific
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single-stranded DNA sequences and are cleaved by the 5′ to 3′ nuclease activity of the DNA polymerase while amplification is occurring. Once the reporter and quencher
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dyes are separated by this cleavage, the fluorescent activity of the reporter dye is unmasked. With each PCR cycle, increasing amounts of cleaved probes are generated
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and the cumulative signal of the reporter dye is concomitantly increased.35,36 Real-
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time detection of PCR products is accomplished by measuring the fluorescence of
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released reporter dyes representing the viral targets.35,36
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Published studies that have used the cobas PCR NAT system
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In a recent study, Müller et al.26 used a commercial, multi-dye nucleic acid
amplification technology test for HBV/HCV and HIV-1/2 to assess the analytical sensitivity of the test in samples containing low concentrations of each virus. The study describes the evaluation of the MPX v2 test by two blood banks in Europe: the German Red Cross Blood Donor Service (Frankfurt, Germany) and the Centro de Hemoterapia y Hemodonacíon de Castilla y León (Valladolid, Spain). The analytical sensitivities of the MPX v2 test determined by the German and Spanish blood banks were 1.1 and 3.5 IU/ml for HBV, 3.9 and 17.6 IU/ml for HCV, and 43.3 and 50.6 IU/ml for HIV-1, respectively.26
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In another study, Roche37 reported on the analytical sensitivities of two versions of the MPX test (MPX v1 and MPX v2) in the detection of HBV, HCV, and HIV-1. In the case of the current commercial NAT test – the MPX v1 test – a reactive
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sample has to be tested further with three individual, virus-specific NAT tests in order to identify the viral contamination. One of the major disadvantages of this strategy is
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that a confirmed reactive sample is often non-reactive when tested with discriminatory tests.38–40 This discrepancy is most likely to occur with samples containing low viral
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loads, typically samples from donors with occult HBV infection.39 The MPX v2 test
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that was evaluated in this study was the first commercial multiplex test able to simultaneously detect and identify viral contaminants in plasma samples. The MPX
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v2 test thus eliminates the drawbacks of the previous version of the test.26 The lower limits of detection (IU/ml) of the MPX v1 test were 3.8, 11.0, and 49.0 for HBV,
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HCV, and HIV-1, respectively, and in the case of the MPX v2 test, the limits were
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2.3, 6.8, and 46.2 for HBV, HCV, and HIV-1, respectively.37,41 Previous studies42,43 found the MPX v1 test to be more sensitive for HBV than for HCV, and although it
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appears that the analytical sensitivity of the Ultrio test is more sensitive than the MPX tests for HIV-1, the two studies that have directly compared the methods43,44 reported
these differences between the Ultrio and MPX v1 tests not to be significant. Similarly, the analytical sensitivities of the German Red Cross kits for HBV and HIV-1 detection appear to be significantly more sensitive than those of the MPX v2 test; however, the limited clinical research comparing the two tests showed no difference in clinical performance.26 The performance of the MPX v2 test for routine donor samples was compared
with the tests on record at the two blood banks.26 In the latter case, an HBV-positive sample was not detected with the test on record, which was the MPX v1 test, although
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the analytical sensitivities of the MPX v1 and MPX v2 tests are very similar (95% limit of detection of 3.8 and 2.3 IU/ml, respectively).26 One explanation for this could be that the donor in this case was a chronic HBV carrier with a low HBV titer that
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was detected intermittently.39 The performance of the MPX v2 test for testing routine donor samples was comparable to both of the in-house tests of the German Red Cross
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Blood donor Service and the MPX v1 test at Centro de Hemoterapia y Hemodonacíon de Castilla y León.26
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Stramer et al.42 [Au?7] provide a summary of HBV DNA analytical
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sensitivities of commercially available triplex NAT assays. These data are limited to those studies using well-characterized WHO stock preparations. HBV DNA analytical
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sensitivity at a 95% limit of detection is approximately 4 IU/ml (range 3–8 IU/ml), and HVC and HIV analytical sensitivities (95% limit of detection) are approximately
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7–22 IU/m and 42–58 IU/ml, respectively.
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Procleix Ultrio assay: TMA
The state-of-the-art TMA technology used in the Procleix Ultrio assay was developed by Gen-Probe, Inc. (San Diego, CA, USA), our partner for NAT innovation. The assay makes use of the Procleix Tigris System, which is a fully automated system for nucleic acid amplification and detection based on TMA technology. The Procleix Ultrio assay procedure consists of three main steps: target
capture, amplification, and detection. During target capture, samples are prepared for testing by virus lysis to release the genetic material. No pretreatment or sample handling is required. Capture probes hybridize with IC and viral nucleic acids and bind them to magnetic particles. Unbound, non-specific material is removed by
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washing to minimize potential inhibitors. RNA and/or DNA amplification in the Procleix Ultrio assay occurs via TMA. Reverse transcriptase creates a DNA copy (cDNA) of the target nucleic acid, and RNA polymerase initiates transcription,
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synthesizing RNA from the cDNA. Some of the newly synthesized RNA amplification products reenter the TMA process and serve as templates for new
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rounds of amplification. Billions of copies can potentially be generated in less than an hour.43 Detection follows on from the amplification step. Acridinium ester (AE)-
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labeled probes specifically hybridize to amplification products. Different AE variants
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are used to label IC- and viral-specific probes. The hybridization protection assay (HPA) process selectively inactivates the AE labels on unhybridized probes to
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minimize background signal, and dual kinetic assay (DKA) technology allows for the simultaneous detection of both IC-encoded and viral-encoded RNA by the detection
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RNA.43
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of a brief flash of light and a longer-lasting glow, respectively, produced by the
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Published literature on TMA-based NATs
A new version of the TMA assay, Ultrio Plus, was recently launched in the European Union (EU), and we were interested to know whether this new version of the assay has improved sensitivity in detecting low viral load HBV compared with the original assay. In comparison to the original Ultrio assay, the Ultrio Plus assay includes an additional reagent that contains concentrated lithium hydroxide, which enhances the disruption of HBV particles and the subsequent release of DNA for the capture probe to target.
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Grabarczyk et al.4 reported on a direct comparison of the two TMA assays for the detection of HBV, HCV, and HIV in blood donors. The analytical sensitivities of the Ultrio and Ultrio Plus assays were analyzed using dilution panels of WHO
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international standards with the following concentrations: WHO HBV genotype A: 50, 15, 5, 1.5, 0.5, 0.15, and 0.05 IU/ml; WHO HCV subtype 1: 100, 30, 10, 3, 1, 0.3,
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and 0.1 IU/ml; and WHO HIV-1 subtype B: 600, 200, 60, 20, 6, 2, and 0.6 IU/ml. A
comparison of the 50% and 95% limits of detection by probit analysis in parallel line
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mode showed the two versions of the TMA assays to have equal analytical sensitivity to the WHO HCV and HIV-1 standards, but in the case of HBV, the Ultrio Plus assay
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was found to have a 2.4-fold higher sensitivity than the Ultrio assay.4 The assessment
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of the analytical sensitivities of the two assays showed the 95% limits of detection for HBV DNA, HCV RNA, and HIV-1 RNA to be 11.1, 9.0, and 14.2 IU/ml,
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Ultrio Plus assay.4
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respectively, for the Ultrio assay, and 4.6, 9.3, and 18.5 IU/ml, respectively, for the
Stramer et al.42 [Au?7] carried out a comparative study of triplex NAT assays
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in the USA. The results of the study showed the 95% limit of detection of HBV DNA to be 10–15 IU/ml (95% range, 8–40 IU/ml) in the Ultrio assay and 2–4 IU/ml (95% range, 2–10 IU/ml) for the Ultrio Plus assay. The Ultrio Plus assay was thus more sensitive for HBV DNA detection than the Ultrio assay. According to studies reported on in the product package inserts, the HCV analytical sensitivities for the two assays are equal (2–6 IU/ml), as are the HIV-1 analytical sensitivities (16–40 IU/ml). In a comparative study evaluating triplex NATs for the detection of HBV
DNA, HCV RNA, and HIV-1 RNA by Xiao et al.,43 the Procleix Tigris System often detected HBV at viral levels below 50 IU/ml. As the HBV viral loads in the chronic
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donations were close to the NAT cut-off (approximately 15 IU/ml for the Procleix Ultrio assay) [Au?8].44
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Discussion
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Fully automated triplex (HIV, HCV, and HBV) NAT assays have been available in
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the USA since 2007, and guidelines regarding the requirements for HIV-1 and HCV NAT assays were issued in 2010.45 In November 2012, the FDA issued guidelines
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regarding HBV DNA detection NAT assays, stipulating that such assays should have a minimum sensitivity of 100 IU/ml (approximately 500 copies/ml).46
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The main advantage of the MPX v2 test over other licensed blood screening NATs currently being used, is its ability to simultaneously detect and identify the viral
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target in a sample. The need for supplementary viral target discriminatory tests is
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eliminated in this assay, as are the associated training requirements. Multiplex, multidye tests also reduce the required sample volumes, which is important in an
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environment in which the number of viral agents being tested for by NATs has gradually increased over the last few years.26 The MPX v2 test is currently limited to
four channels and dyes, one of which is used for the IC. Consequently, all HIV viruses (HIV-1 groups M and O and HIV-2) are detected in a single channel, and thus further testing is required.26 The Roche MPX test carried out on the s 201 platform represents an improved and fully automated NAT blood and plasma screening system.26 As shown in our results, this technology proved to be highly sensitive
compared with the technologies developed in-house in our group.17,20 The analytical sensitivity studies on the Ultrio and Ultrio Plus assay systems showed that the two TMA assays are equally sensitive for the detection of HCV and
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HIV-1. However, the inclusion of an alkaline shock step to the virus particle during the target capture process has significantly improved the analytical sensitivity of the assay to HBV in the Ultrio Plus assay. The NAT efficiency in both TMA assay
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versions appears to be optimal for HCV and HIV-1. The detection efficiency of the original Ultrio assay for HBV DNA, however, was poor, and ranged from 2% to 43%.
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This range was increased to 18% to 81% by improved target capture chemistry in the Ultrio Plus assay.4 One explanation for this is that the improvement offered by the
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alkaline shock depends largely on the length of the double-stranded (ds) DNA portion
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of the HBV genome.4 This dsDNA portion varies even within one carrier,47 and may be even more variable between individuals infected with different HBV variants. It is
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thus possible that denaturation of dsDNA is an additional contributing factor to the enhanced sensitivity of the Ultrio Plus assay, and that the remaining variability in
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limits of detection in the Ultrio Plus assay is a reflection of incomplete denaturation of
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dsDNA during target capture. Another explanation for the higher detection efficiency of the Ultrio Plus assay for HBV is the denaturation of a tightly attached protein to the
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HBV genome47 that otherwise could inhibit target capture by steric hindrance. Such
an inhibitory effect by interfering proteins does not play a role in capturing HIV RNA and HCV RNA, which may explain why the NAT efficiencies for these viruses are
found to be higher than that for HBV. Regardless of the explanation, the alkaline shock step has certainly contributed to an enhanced sensitivity in the Ultrio Plus assay.
The results of a comparative study of the Ultrio and Ultrio Plus assays showed that there was no difference in the analytical sensitivities of the two assays to HCV and HIV-1, but that the Ultrio Plus assay had a three-fold higher sensitivity to HBV than the Ultrio assay.
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Conclusions
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Although the introduction of real-time PCR has led to considerable progress in automating the amplification and detection steps of NATs, nucleic acid isolation
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remains very labor-intensive when performed manually. Traditional phenol–
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chloroform extraction and ethanol precipitation methods are complicated, timeconsuming, hazardous, and unsuitable for processing high numbers of samples.
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Some technical problems of ‘in-house’ nucleic acid amplification technology for detection purposes are recognized widely, especially: (1) manual extraction of
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nucleic acids, enabling the loss of RNA or DNA; (2) low sensitivity of the amplification reaction; (3) optimal amplification design and thorough optimization of
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the amplification conditions; (4) a reproducible quantitative amplification assay is
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very difficult to construct and it remains laborious and cumbersome. Thus, the automation of nucleic acid extraction and amplification technology becomes
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necessary.
Acknowledgements
This work was supported by grants from Fundação de Amparo a Pesquisa do Estado de São Paulo (FAPESP), Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), Coordenação de Aperfeiçoamento de Pessoal de Nível superior (CAPES), Fundação Oswaldo Ramos (FOR), and Fundo de Auxílio aos Docentes e Alunos (FADA). The funders had no role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript.
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Conflict of interest: The authors have declared that no competing interests exist.
Tabor E, Epstein JS. NAT screening of blood and plasma donations: evolution
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Glaubitz J, Sizmann D, Simon CO, et al. [Au?9] Accuracy to 2nd International HIV-1 RNA WHO Standard: assessment of three generations of quantitative HIV-1 RNA nucleic acid amplification tests. J Clin Virol 2011;50:119–24.
22.
De Crignis E, Re MC, Cimatti L, et al. [Au?9] HIV-1 and HCV detection in dried blood spots by SYBR Green multiplex real-time RT-PCR. J Virol Methods 2010;165:51–6.
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23.
Oliveira dos Santos A, Souza LF, Borzacov LM, et al. [Au?9] Development of cost-effective real-time PCR test: to detect a wide range of HBV DNA concentrations in the western Amazon region of Brazil. Virol J 2014;11:16. Yalamanchili N, Syed R, Chandra M, et al. [Au?9] A latest and promising
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24.
approach for prediction of viral load in hepatitis B virus infected patients.
25.
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Indian J Hum Genet 2011;17:17–21.
Takizawa K, Nakashima T, Mizukami T, et al. [Au?9] Degenerate polymerase
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chain reaction strategy with DNA microarray for detection of multiple and
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various subtypes of virus during blood screening. Transfusion 2013;53:2545– 55.
Müller MM, Fraile MI, Hourfar MK, et al. [Au?9] Evaluation of two
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commercial, multi-dye, nucleic acid amplification technology tests, for
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HBV/HCV/HIV-1/HIV-2 and B19V/HAV, for screening blood and plasma for
27.
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further manufacture. Vox Sang 2013;104:19–29. Roche Molecular Systems, Inc. cobas® TaqScreen MPX® Test for use on the
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cobas® s 201 System®. Product insert: 1636, Rev. 4. Indianapolis, IN: Roche; 2009. [Au?10]
28.
Gen-Probe Inc. Procleix® Ultrio® Assay. Product insert: 502165, Rev. A. San Diego, CA: Gen-Probe Inc.; 2011.
29.
Gen-Probe Inc. Procleix® Ultrio® Plus Assay. Product insert: 502932, Rev. 7. San Diego, CA: Gen-Probe Inc.; 2012.
30.
Stramer SL, Zou S, Notari EP, et al. [Au?9] Blood donation screening for hepatitis B virus markers in the era of nucleic acid testing: are all tests of value? Transfusion 2012;52:440–6.
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31.
cobas® TaqScreen MPX® Test for use on the cobas® s201 System®. Roche; 2009. Available at: http://www.fda.gov/downloads/.../UCM176443.pdf (accessed [Au?11]). [Au?10] Longo MC, Berninger MS, Hartley JL. Use of uracil DNA glycosylase to
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control carry-over contamination in polymerase chain reactions. Gene
33.
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Savva R, McAuley-Hecht K, Brown T, et al. [Au?9] The structural basis of
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specific base-excision repair by uracil-DNA glycosylase. Nature
34.
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1995;373:487–93.
Mol CD, Arvai AS, Slupphaug G, et al. [Au?9] Crystal structure and
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mutational analysis of human uracil-DNA glycosylase: structural basis for specificity and catalysis. Cell 1995;80:869–78. Higuchi R, Dollinger G, Walsh PS, et al. [Au?9] Simultaneous amplification
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35.
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Heid CA, Stevens J, Livak KJ, et al. [Au?9] Real time quantitative PCR.
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36.
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and detection of specific DNA sequences. Biotechnology (N Y) 1992;10:413–
Genome Res 1996;6:986–94.
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cobas® TaqScreen MPX Test, v2.0 for use on the cobas® s 201 System. Rev. 1, 01/2011. Package insert. Branchburg, NJ: Roche Molecular Systems, Inc.; 2011.
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Brojer E, Grabarczyk P, Liszewski G, et al. [Au?9] Characterization of HBV DNA+/HBsAg− blood donors in Poland identified by triplex NAT. Hepatology 2006;44:1666–74.
39.
Li L, Chen PJ, Chen MH, et al. [Au?9] A pilot study for screening blood donors in Taiwan by nucleic acid amplification technology: detecting occult
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hepatitis B virus infections and closing the serologic window period for hepatitis C virus. Transfusion 2008;48:1198–206. 40.
Dettori S, Candido A, Kondili LA, et al. [Au9?] Identification of low HBV-
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DNA levels by nucleic acid amplification test (NAT) in blood donors. J Infect 2009;59:128–33.
cobas® TaqScreen MPX® Test, for use on the cobas® s201 System®, Rev. 5,
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01/2010. Package insert. Branchburg, NJ: Roche Molecular Systems, Inc.;
Assal A, Barlet V, Deschaseaux M, et al. [Au?9] Sensitivity of two hepatitis B
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2010.
virus, hepatitis C virus (HCV), and human immunodeficiency virus (HIV)
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nucleic acid test systems relative to hepatitis B surface antigen, anti-HCV, anti-HIV, and p24/anti-HIV combination assays in seroconversion panels.
Xiao X, Zhai J, Zeng J, et al. [Au?9] Comparative evaluation of a triplex
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Transfusion 2009;49:301–10.
nucleic acid test for detection of HBV DNA, HCV RNA, and HIV-1 RNA,
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with the Procleix Tigris System. J Virol Methods 2013;187:357–61.
44.
McCormick MK, Dockter J, Linnen JM, et al. [Au?9] Evaluation of a new molecular assay for detection of human immunodeficiency virus type 1 RNA, hepatitis C virus RNA, and hepatitis B virus DNA. J Clin Virol 2006;36:166– 76.
45.
US Food and Drug Administration. Guidance for industry: Nucleic acid testing (NAT) for human immunodeficiency virus type 1 (HIV-1) and hepatitis C virus (HCV): testing, product disposition, and donor deferral and reentry. US FDA; [Au?12]. Available at:
Page 22 of 27
http://www.fda.govQBiologicsBloodVaccinesQGuidanceComplianceRegulato ryInformation/Guidances/default.htm (accessed [Au?11]). 46.
US Food and Drug Administration. Guidance for industry: use of nucleic acid
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tests on pooled and individual samples from donors of whole blood and blood
hepatitis B virus. US FDA; [Au?12]. Available at:
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components, including source plasma, to reduce the risk of transmission of
http://www.fda.gov/BiologicsBloodVaccines/GuidanceComplianceRegulatory
Landers TA, Greenberg HB, Robinson WS. Structure of hepatitis B Dane
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Information/Guidances/default.htm (accessed [Au?11]).
particle DNA and nature of the endogenous DNA polymerase reaction. J Virol
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te
d
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1977;23:368–76.
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Figure captions
Figure 1 Standard curve of the HIV RNA concentration (IU/ml) (slope: −3.20; R2:
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0.99).
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Figure 2 Standard curve of the HBV DNA concentration (IU/ml) (slope: −2.75; R2:
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te
d
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an
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0.98).
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Figure 1
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Figure 2
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Highlights - We describe current molecular methods for the detection of HIV, HCV, and HBV.
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- We compared the lower limit of virus detection and evaluated the overall performance.
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- In house PCR the limit of detection for HCV/HBV/HIV was 310;200;100
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UI/mL respectivelly.
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S1201-9712(14)01502-1 http://dx.doi.org/doi:10.1016/j.ijid.2014.04.007 IJID 1979
To appear in:
International Journal of Infectious Diseases
Received date: Revised date: Accepted date:
6-3-2014 1-4-2014 9-4-2014
Please cite this article as: Guilherme AlbertoniManoel Jo˜ao Batista Castelo Gir˜aoNestor Schor Mini review:Current molecular methods for the detection and quantification of hepatitis B virus, hepatitis C virus, and human immunodeficiency virus type 1 (2014), http://dx.doi.org/10.1016/j.ijid.2014.04.007 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Running heads: Recto: Molecular methods for detection of viruses
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Verso: G. Albertoni et al.
Correspondence footnote:
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[ARTICLE TYPE – REVIEW ARTICLE]
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E-mail address: [email protected] (G. Albertoni).
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*Corresponding author. Tel.: +55 11 993647519; fax: +55 11 50556588.
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Mini review: Current molecular methods for the detection and quantification of hepatitis B virus, hepatitis C virus, and
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human immunodeficiency virus type 1
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Guilherme Albertonia,b,*, Manoel João Batista Castelo Girãob, Nestor Schora
a
Federal University of São Paulo (UNIFESP), Department of Medicine, Nephrology
Division, Rua Botucatu 740, São Paulo, 04023-900, SP, Brazil b
Colsan (Associação Beneficente de Coleta de Sangue), São Paulo, SP, Brazil
[Received date] Corresponding Editor: Larry Lutwick, Kalamazoo, Michigan, USA
KEYWORDS
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NAT testing; HIV; HCV;
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HBV
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Summary
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The detection of acute human immunodeficiency virus (HIV), hepatitis B virus
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(HBV), and hepatitis C virus (HCV) infection is vital for controlling the spread of HIV, HBV, and HCV to uninfected individuals. Considering that these viruses have
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high replication rates and are undetectable by serological markers, early detection upon transmission is crucial. Various nucleic acid assays have been developed for
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diagnostics and therapeutic monitoring of infections. In the past decade, rapid and
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sensitive molecular techniques such as PCR have revolutionized the detection of a variety of infectious viruses, including HIV, HCV, and HBV. Here, we describe two
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of the most commonly used licensed methods for the detection and quantification of HIV, HCV, and HBV: the cobas TaqScreen MPX (PCR) test and the Tigris System. We used transcription-mediated amplification to review and compare the development and efficiency of these technologies.
Introduction
The quality and safety of blood products used in transfusions are major public health concerns. In addition to general quality control (QC), the introduction of good manufacturing practices and routine screening of blood materials and products, has
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ensured consistency, quality, and safety in the increased production and use of blood products in recent decades. Newly developed serological tests and nucleic acid tests (NATs) have markedly reduced the risk of transmission of human immunodeficiency
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virus (HIV), hepatitis C virus (HCV), and hepatitis B virus (HBV).1 In the past decade, rapid and sensitive molecular techniques such as PCR have revolutionized the
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detection of a variety of infectious viruses.2–4
About 10 years ago, molecular methods achieved a significant level of
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throughput and automation, enabling their introduction as an additional tool in blood screening. The aim of these tests was primarily to prevent donations made during the
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window-period from being used in transfusions.5,6 An international survey7 reported
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findings from the use of NATs in 330 million blood donations and showed overall infection rates of 1:447 000 for HCV, 1:111 000 for HIV, and 1:66 000 for HBV. Up
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until 2011, very few of Brazil’s blood banks (corresponding to less than 5% of the
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national blood supply) had voluntarily introduced NAT screening. More recently, an HCV/HIV NAT kit was developed by Bio-Manguinhos/Fiocruz (Rio de Janeiro,
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Brazil). This test is now used in some of Brazil’s large public blood banks and is expected to be extended to all public blood banks by 2012 when the test may become mandatory [Au?1].8
In this article, we describe two of the most commonly used licensed molecular
methods for the detection and quantification of HIV, HCV, and HBV in blood. We compared the lower limits of virus detection of each method and evaluated the overall performance.
Nucleic acid testing (NAT)
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The World Health Organization (WHO) recommends that diagnostic devices are ‘ASSURED’: Affordable, Sensitive, Specific, User-friendly, Rapid and Robust, Equipment-free, and Deliverable to end users, especially with regard to developing
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countries. Nucleic acid amplification techniques (NAATs), however, typically require a significant investment in equipment, training, and infrastructure. Nevertheless, the
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development of these molecular techniques as diagnostic tools has become
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increasingly important.9–13
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Real-time PCR
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The fact that PCR reactions can be monitored in real time has revolutionized the process of DNA and RNA fragment quantification. Nucleic acid quantification using
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quantitative real-time PCR (qRT-PCR) is extremely accurate and reproducible. In
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qRT-PCR, each round of amplification leads to the emission of a fluorescent signal, and the number of signals per cycle is proportional to the amount of target nucleic
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acid in the starting sample.16 [Au?2] This allows for accurate and reproducible quantification based on fluorescence. The most commonly used fluorescent compounds in qRT-PCR are the SYBR Green dye and the TaqMan probe.16 qRT-PCR
requires an instrumentation platform that consists of a thermal cycler, a computer, optics for fluorescence excitation and emission collection, and data acquisition and analysis software.14–16
In-house NAT for HCV detection
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For the detection and quantification of HCV RNA,17 we designed and developed a one-step NAT TaqMan qRT-PCR method. Using international standards (ACCURUN HCV RNA Positive Control;
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SeraCare) containing 3 × 105 IU/ml of HCV RNA (1 U = 1–3 copies),14 our NAT inhouse qRT-PCR TaqMan HCV assay displayed linearity over a range of 3.1 × 102 to
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3.1 × 105 IU/ml (2.5–5.5 log). The lower limit of detection of the method was 310
IU/ml. In a previous report by Levi et al.,18 [Au?3] the method used to screen blood
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donors by NAT testing for HCV RNA displayed linearity over a range of 5 × 102 to 5 × 104. The lower limit of detection of the method was 500 IU/ml. In another study,
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Paryan et al.19 designed and developed an in-house multiplex RT-PCR assay for the
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detection of HCV in plasma samples. That multiplex assay was linear between 102 and 105 copies/ml (six of eight samples were detectable), and had an analytical
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sensitivity of 200 copies/ml.
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In-house NAT for HIV detection
We designed and developed a one-step TaqMan NAT qRT-PCR method for the detection of HIV RNA.20
We recently developed a NAT TaqMan qRT-PCR HIV RNA assay using a set
of international standards (AcroMetrix HIV-1 Panel IU/ml RNA Positive Control; Applied Biosystems) containing 1 × 102 to 1 × 107 IU/ml of HIV RNA. Each panel member (except the negative control) contains HIV-1 at a predetermined concentration calibrated against the WHO international standard for HIV-1 RNA. Consequently, the assigned values are reported in international units per milliliter (IU/ml). Some studies have reported results in units that differ from the international
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units as defined by the WHO.21 For our in-house NAT TaqMan qRT-PCR HIV assay, we observed linearity over a range of 1 × 102 to 1 × 107 IU/ml and a lower limit of detection of 100 IU/ml (Figure 1). Similarly, Paryan et al.,19 using multiplex RT-PCR,
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showed linearity over a range of 1 × 102 to 1 × 105 copies/ml and an analytical sensitivity of 100 copies/ml for HIV RNA. In experiments by De Cringes et al.,22
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[Au?4] a SYBR Green I Multiplex RT-PCR was successfully used to detect HIV
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RNA, and was found to have an analytical sensitivity of 400 copies/ml for HIV RNA.
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[Figure 1 here]
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In-house NAT for HBV detection
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We developed a NAT in-house qRT-PCR TaqMan HBV DNA assay using
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international standards (AcroMetrix HBV Panel IU/ml DNA Positive Control; Applied Biosystems) containing 2 × 102 to 2 × 107 IU/ml of HBV DNA. Our HBV
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assay displayed linearity over a range of 2 × 102 to 2 × 107 IU/ml, and the lower limit of detection of the method was 200 IU/ml (Figure 2 [Au?5]). dos Santos et al.23 developed a cost-effective RT-PCR test to detect a wide range of HBV DNA in the western Amazonas region. Their method displayed linearity over a range of 2 × 103 to 2 × 109 copies/ml and a lower limit of detection of 2000 IU/ml. Yalamanchili et al.24
designed a rapid, reliable, and sensitive assay for the detection of HBV by RT-PCR. The calibration curve was linear over a range of 1 × 102 to 1 × 108 copies/ml, with an
R2 value of 0.999. The lower limit of detection of the method was 100 copies/ml.
[Figure 2 here]
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Current approaches
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Several US Food and Drug Administration (FDA)-licensed NAT assays are currently available for the screening of blood donors for HIV, HCV, HBV, and West Nile virus
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(WNV). The continued development of highly sensitive screening NAT systems,
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however, is challenging.25 The FDA requires that blood screening has licensed several triplex (HIV/HCV/HBV) automated NAT systems for blood donor screening
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[Au?6].26 These include the PCR-based cobas TaqScreen MPX assay using the cobas s 201 instrument (Roche Diagnostics GmbH, Mannheim, Germany), and the Procleix
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Ultrio assay, using the Procleix Tigris automated instrument (Novartis Diagnostics, Emeryville, CA, USA/Gen-Probe, San Diego, CA, USA) and employing
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transcription-mediated amplification (TMA).27–30
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PCR-based cobas TaqScreen MPX test
The cobas TaqScreen MPX test is a multiplex, multi-dye nucleic acid amplification technology. Sample nucleic acids and armored RNA internal control (IC) molecules, which serve as specimen preparation and amplification/detection process controls, are processed simultaneously. A protease solution digests proteins to promote lysis, inactivate nucleases, and facilitate the release of RNA and DNA from viral particles. The released nucleic acids bind to the silica surface of magnetic glass particles, which are added to the reaction mixture. The binding of nucleic acids to the particles is mainly due to the net positive charge on the glass particle surface and net negative charge of the nucleic acids at the chaotropic salt concentration and ionic strength of
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the lysis reaction. A wash reagent removes unbound substances and impurities. Using an elution buffer, purified nucleic acids are then released from the magnetic glass particles at high temperatures.31
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After isolation of the purified nucleic acids from human plasma, cobas TaqScreen Master Mix (MPX MMX) is used for the amplification and detection of
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HIV-1 (groups M and O), HIV-2, and HCV RNA, as well as HBV DNA and IC RNA. Once the MPX MMX is activated by the addition of manganese acetate, reverse
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transcription (for RNA targets) proceeds, followed by PCR amplification of highly conserved regions of HIV-1 (groups M and O), HIV-2, and HCV RNA, as well as
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HBV DNA and IC RNA using specific primers.31
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Simultaneous detection of the amplified nucleic acids is accomplished by the generation of fluorescent signals from 5′-nucleolytic degradation of HIV-1- (groups
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M and O), HIV-2-, HCV-, HBV- and IC-specific probes. Two fluorescent dyes are
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used, one labeling the IC probe and a second labeling all target-specific probes, allowing for the combined identification of each viral target as well as independent
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identification of the IC. Reverse transcription and amplification reactions are performed with a thermostable recombinant DNA polymerase enzyme. In the presence of manganese (Mn2+), DNA polymerase has reverse transcriptase and DNA polymerase activities. This allows both reverse transcription and PCR amplification to occur in the same reaction mixture.31 Selective amplification of target nucleic acid from the specimen is achieved by
the use of a uracil-N-glycosylase enzyme and deoxyuridine triphosphate. The enzyme recognizes and catalyzes the destruction of DNA strands containing deoxyuridine,32 but not DNA containing deoxythymidine or RNA containing ribouridine.33,34
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During PCR amplification, the intermittent high temperatures during thermal cycling denature target and IC amplicons to form single-stranded DNA. The specific oligonucleotide probes, which facilitate signal detection, hybridize to the single-
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stranded form of the amplified DNA, and amplification, hybridization, and detection occur simultaneously.33,35,36 During PCR amplification, probes hybridize to specific
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single-stranded DNA sequences and are cleaved by the 5′ to 3′ nuclease activity of the DNA polymerase while amplification is occurring. Once the reporter and quencher
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dyes are separated by this cleavage, the fluorescent activity of the reporter dye is unmasked. With each PCR cycle, increasing amounts of cleaved probes are generated
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and the cumulative signal of the reporter dye is concomitantly increased.35,36 Real-
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time detection of PCR products is accomplished by measuring the fluorescence of
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released reporter dyes representing the viral targets.35,36
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Published studies that have used the cobas PCR NAT system
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In a recent study, Müller et al.26 used a commercial, multi-dye nucleic acid
amplification technology test for HBV/HCV and HIV-1/2 to assess the analytical sensitivity of the test in samples containing low concentrations of each virus. The study describes the evaluation of the MPX v2 test by two blood banks in Europe: the German Red Cross Blood Donor Service (Frankfurt, Germany) and the Centro de Hemoterapia y Hemodonacíon de Castilla y León (Valladolid, Spain). The analytical sensitivities of the MPX v2 test determined by the German and Spanish blood banks were 1.1 and 3.5 IU/ml for HBV, 3.9 and 17.6 IU/ml for HCV, and 43.3 and 50.6 IU/ml for HIV-1, respectively.26
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In another study, Roche37 reported on the analytical sensitivities of two versions of the MPX test (MPX v1 and MPX v2) in the detection of HBV, HCV, and HIV-1. In the case of the current commercial NAT test – the MPX v1 test – a reactive
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sample has to be tested further with three individual, virus-specific NAT tests in order to identify the viral contamination. One of the major disadvantages of this strategy is
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that a confirmed reactive sample is often non-reactive when tested with discriminatory tests.38–40 This discrepancy is most likely to occur with samples containing low viral
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loads, typically samples from donors with occult HBV infection.39 The MPX v2 test
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that was evaluated in this study was the first commercial multiplex test able to simultaneously detect and identify viral contaminants in plasma samples. The MPX
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v2 test thus eliminates the drawbacks of the previous version of the test.26 The lower limits of detection (IU/ml) of the MPX v1 test were 3.8, 11.0, and 49.0 for HBV,
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HCV, and HIV-1, respectively, and in the case of the MPX v2 test, the limits were
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2.3, 6.8, and 46.2 for HBV, HCV, and HIV-1, respectively.37,41 Previous studies42,43 found the MPX v1 test to be more sensitive for HBV than for HCV, and although it
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appears that the analytical sensitivity of the Ultrio test is more sensitive than the MPX tests for HIV-1, the two studies that have directly compared the methods43,44 reported
these differences between the Ultrio and MPX v1 tests not to be significant. Similarly, the analytical sensitivities of the German Red Cross kits for HBV and HIV-1 detection appear to be significantly more sensitive than those of the MPX v2 test; however, the limited clinical research comparing the two tests showed no difference in clinical performance.26 The performance of the MPX v2 test for routine donor samples was compared
with the tests on record at the two blood banks.26 In the latter case, an HBV-positive sample was not detected with the test on record, which was the MPX v1 test, although
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the analytical sensitivities of the MPX v1 and MPX v2 tests are very similar (95% limit of detection of 3.8 and 2.3 IU/ml, respectively).26 One explanation for this could be that the donor in this case was a chronic HBV carrier with a low HBV titer that
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was detected intermittently.39 The performance of the MPX v2 test for testing routine donor samples was comparable to both of the in-house tests of the German Red Cross
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Blood donor Service and the MPX v1 test at Centro de Hemoterapia y Hemodonacíon de Castilla y León.26
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Stramer et al.42 [Au?7] provide a summary of HBV DNA analytical
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sensitivities of commercially available triplex NAT assays. These data are limited to those studies using well-characterized WHO stock preparations. HBV DNA analytical
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sensitivity at a 95% limit of detection is approximately 4 IU/ml (range 3–8 IU/ml), and HVC and HIV analytical sensitivities (95% limit of detection) are approximately
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7–22 IU/m and 42–58 IU/ml, respectively.
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Procleix Ultrio assay: TMA
The state-of-the-art TMA technology used in the Procleix Ultrio assay was developed by Gen-Probe, Inc. (San Diego, CA, USA), our partner for NAT innovation. The assay makes use of the Procleix Tigris System, which is a fully automated system for nucleic acid amplification and detection based on TMA technology. The Procleix Ultrio assay procedure consists of three main steps: target
capture, amplification, and detection. During target capture, samples are prepared for testing by virus lysis to release the genetic material. No pretreatment or sample handling is required. Capture probes hybridize with IC and viral nucleic acids and bind them to magnetic particles. Unbound, non-specific material is removed by
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washing to minimize potential inhibitors. RNA and/or DNA amplification in the Procleix Ultrio assay occurs via TMA. Reverse transcriptase creates a DNA copy (cDNA) of the target nucleic acid, and RNA polymerase initiates transcription,
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synthesizing RNA from the cDNA. Some of the newly synthesized RNA amplification products reenter the TMA process and serve as templates for new
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rounds of amplification. Billions of copies can potentially be generated in less than an hour.43 Detection follows on from the amplification step. Acridinium ester (AE)-
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labeled probes specifically hybridize to amplification products. Different AE variants
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are used to label IC- and viral-specific probes. The hybridization protection assay (HPA) process selectively inactivates the AE labels on unhybridized probes to
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minimize background signal, and dual kinetic assay (DKA) technology allows for the simultaneous detection of both IC-encoded and viral-encoded RNA by the detection
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RNA.43
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of a brief flash of light and a longer-lasting glow, respectively, produced by the
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Published literature on TMA-based NATs
A new version of the TMA assay, Ultrio Plus, was recently launched in the European Union (EU), and we were interested to know whether this new version of the assay has improved sensitivity in detecting low viral load HBV compared with the original assay. In comparison to the original Ultrio assay, the Ultrio Plus assay includes an additional reagent that contains concentrated lithium hydroxide, which enhances the disruption of HBV particles and the subsequent release of DNA for the capture probe to target.
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Grabarczyk et al.4 reported on a direct comparison of the two TMA assays for the detection of HBV, HCV, and HIV in blood donors. The analytical sensitivities of the Ultrio and Ultrio Plus assays were analyzed using dilution panels of WHO
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international standards with the following concentrations: WHO HBV genotype A: 50, 15, 5, 1.5, 0.5, 0.15, and 0.05 IU/ml; WHO HCV subtype 1: 100, 30, 10, 3, 1, 0.3,
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and 0.1 IU/ml; and WHO HIV-1 subtype B: 600, 200, 60, 20, 6, 2, and 0.6 IU/ml. A
comparison of the 50% and 95% limits of detection by probit analysis in parallel line
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mode showed the two versions of the TMA assays to have equal analytical sensitivity to the WHO HCV and HIV-1 standards, but in the case of HBV, the Ultrio Plus assay
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was found to have a 2.4-fold higher sensitivity than the Ultrio assay.4 The assessment
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of the analytical sensitivities of the two assays showed the 95% limits of detection for HBV DNA, HCV RNA, and HIV-1 RNA to be 11.1, 9.0, and 14.2 IU/ml,
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Ultrio Plus assay.4
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respectively, for the Ultrio assay, and 4.6, 9.3, and 18.5 IU/ml, respectively, for the
Stramer et al.42 [Au?7] carried out a comparative study of triplex NAT assays
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in the USA. The results of the study showed the 95% limit of detection of HBV DNA to be 10–15 IU/ml (95% range, 8–40 IU/ml) in the Ultrio assay and 2–4 IU/ml (95% range, 2–10 IU/ml) for the Ultrio Plus assay. The Ultrio Plus assay was thus more sensitive for HBV DNA detection than the Ultrio assay. According to studies reported on in the product package inserts, the HCV analytical sensitivities for the two assays are equal (2–6 IU/ml), as are the HIV-1 analytical sensitivities (16–40 IU/ml). In a comparative study evaluating triplex NATs for the detection of HBV
DNA, HCV RNA, and HIV-1 RNA by Xiao et al.,43 the Procleix Tigris System often detected HBV at viral levels below 50 IU/ml. As the HBV viral loads in the chronic
Page 13 of 27
donations were close to the NAT cut-off (approximately 15 IU/ml for the Procleix Ultrio assay) [Au?8].44
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Discussion
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Fully automated triplex (HIV, HCV, and HBV) NAT assays have been available in
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the USA since 2007, and guidelines regarding the requirements for HIV-1 and HCV NAT assays were issued in 2010.45 In November 2012, the FDA issued guidelines
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regarding HBV DNA detection NAT assays, stipulating that such assays should have a minimum sensitivity of 100 IU/ml (approximately 500 copies/ml).46
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The main advantage of the MPX v2 test over other licensed blood screening NATs currently being used, is its ability to simultaneously detect and identify the viral
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target in a sample. The need for supplementary viral target discriminatory tests is
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eliminated in this assay, as are the associated training requirements. Multiplex, multidye tests also reduce the required sample volumes, which is important in an
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environment in which the number of viral agents being tested for by NATs has gradually increased over the last few years.26 The MPX v2 test is currently limited to
four channels and dyes, one of which is used for the IC. Consequently, all HIV viruses (HIV-1 groups M and O and HIV-2) are detected in a single channel, and thus further testing is required.26 The Roche MPX test carried out on the s 201 platform represents an improved and fully automated NAT blood and plasma screening system.26 As shown in our results, this technology proved to be highly sensitive
compared with the technologies developed in-house in our group.17,20 The analytical sensitivity studies on the Ultrio and Ultrio Plus assay systems showed that the two TMA assays are equally sensitive for the detection of HCV and
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HIV-1. However, the inclusion of an alkaline shock step to the virus particle during the target capture process has significantly improved the analytical sensitivity of the assay to HBV in the Ultrio Plus assay. The NAT efficiency in both TMA assay
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versions appears to be optimal for HCV and HIV-1. The detection efficiency of the original Ultrio assay for HBV DNA, however, was poor, and ranged from 2% to 43%.
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This range was increased to 18% to 81% by improved target capture chemistry in the Ultrio Plus assay.4 One explanation for this is that the improvement offered by the
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alkaline shock depends largely on the length of the double-stranded (ds) DNA portion
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of the HBV genome.4 This dsDNA portion varies even within one carrier,47 and may be even more variable between individuals infected with different HBV variants. It is
M
thus possible that denaturation of dsDNA is an additional contributing factor to the enhanced sensitivity of the Ultrio Plus assay, and that the remaining variability in
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limits of detection in the Ultrio Plus assay is a reflection of incomplete denaturation of
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dsDNA during target capture. Another explanation for the higher detection efficiency of the Ultrio Plus assay for HBV is the denaturation of a tightly attached protein to the
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HBV genome47 that otherwise could inhibit target capture by steric hindrance. Such
an inhibitory effect by interfering proteins does not play a role in capturing HIV RNA and HCV RNA, which may explain why the NAT efficiencies for these viruses are
found to be higher than that for HBV. Regardless of the explanation, the alkaline shock step has certainly contributed to an enhanced sensitivity in the Ultrio Plus assay.
The results of a comparative study of the Ultrio and Ultrio Plus assays showed that there was no difference in the analytical sensitivities of the two assays to HCV and HIV-1, but that the Ultrio Plus assay had a three-fold higher sensitivity to HBV than the Ultrio assay.
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Conclusions
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Although the introduction of real-time PCR has led to considerable progress in automating the amplification and detection steps of NATs, nucleic acid isolation
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remains very labor-intensive when performed manually. Traditional phenol–
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chloroform extraction and ethanol precipitation methods are complicated, timeconsuming, hazardous, and unsuitable for processing high numbers of samples.
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Some technical problems of ‘in-house’ nucleic acid amplification technology for detection purposes are recognized widely, especially: (1) manual extraction of
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nucleic acids, enabling the loss of RNA or DNA; (2) low sensitivity of the amplification reaction; (3) optimal amplification design and thorough optimization of
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the amplification conditions; (4) a reproducible quantitative amplification assay is
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very difficult to construct and it remains laborious and cumbersome. Thus, the automation of nucleic acid extraction and amplification technology becomes
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necessary.
Acknowledgements
This work was supported by grants from Fundação de Amparo a Pesquisa do Estado de São Paulo (FAPESP), Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), Coordenação de Aperfeiçoamento de Pessoal de Nível superior (CAPES), Fundação Oswaldo Ramos (FOR), and Fundo de Auxílio aos Docentes e Alunos (FADA). The funders had no role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript.
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Conflict of interest: The authors have declared that no competing interests exist.
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Figure captions
Figure 1 Standard curve of the HIV RNA concentration (IU/ml) (slope: −3.20; R2:
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0.99).
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Figure 2 Standard curve of the HBV DNA concentration (IU/ml) (slope: −2.75; R2:
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0.98).
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Figure 1
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Figure 2
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Highlights - We describe current molecular methods for the detection of HIV, HCV, and HBV.
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- We compared the lower limit of virus detection and evaluated the overall performance.
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- In house PCR the limit of detection for HCV/HBV/HIV was 310;200;100
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UI/mL respectivelly.
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