Primary screening of blood donors by nat testing for HCV-RNA: development of an "in-house" method and results

Wendel, Silvano; Levi, José Eduardo; Takaoka, Deise Tihe; Silva, Isabela Cristina; Castro, Juliana P.; Torezan-Filho, Mário A.; Ghaname, Jorge; Gioachini, Romualdo; Brandão, Joselito; Durigon, Edison Luíz

doi:10.1590/s0036-46652007000300008

Cited by 16 publications

(17 citation statements)

References 29 publications

(31 reference statements)

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“…Genomic screening can be performed on individual donations or in plasma pools ranging between 6 and 96, although it was shown that pooling reduces significantly the yield of DNA‐containing donations 4,5 . In Brazil, despite relatively high prevalence of the marker, anti‐HBc screening is mandatory and a few blood banks also routinely test blood donations for both hepatitis C virus (HCV) and human immunodeficiency virus (HIV) RNA but not for HBV DNA 6 . A fundamental limitation of anti‐HBc screening is the inability to detect window‐period, highly infectious, donations.…”

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confidence: 99%

A probable case of hepatitis B virus transfusion transmission revealed after a 13‐month‐long window period

et al. 2008

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A probable case of hepatitis B virus transfusion transmission revealed after a 13‐month‐long window period

et al. 2008

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“…Albertoni et al (2010) reported a limit of detection for an one-step RT-PCR for HCV detection as 310 IU/mL. Wendel et al (2007) reported a detection limit of 500 IU/mL for their in-house HCV real-time RT-PCR test. Those two works did not use any IC.…”

Section: Discussionmentioning

confidence: 99%

Internal control for real-time polymerase chain reaction based on MS2 bacteriophage for RNA viruses diagnostics

Zambenedetti

Pavoni

Dallabona

et al. 2017

Mem. Inst. Oswaldo Cruz

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BACKGROUND Real-time reverse transcription polymerase chain reaction (RT-PCR) is routinely used to detect viral infections. In Brazil, it is mandatory the use of nucleic acid tests to detect hepatitis C virus (HCV), hepatitis B virus and human immunodeficiency virus in blood banks because of the immunological window. The use of an internal control (IC) is necessary to differentiate the true negative results from those consequent from a failure in some step of the nucleic acid test.OBJECTIVES The aim of this study was the construction of virus-modified particles, based on MS2 bacteriophage, to be used as IC for the diagnosis of RNA viruses.METHODS The MS2 genome was cloned into the pET47b(+) plasmid, generating pET47b(+)-MS2. MS2-like particles were produced through the synthesis of MS2 RNA genome by T7 RNA polymerase. These particles were used as non-competitive IC in assays for RNA virus diagnostics. In addition, a competitive control for HCV diagnosis was developed by cloning a mutated HCV sequence into the MS2 replicase gene of pET47b(+)-MS2, which produces a non-propagating MS2 particle. The utility of MS2-like particles as IC was evaluated in a one-step format multiplex real-time RT-PCR for HCV detection.FINDINGS We demonstrated that both competitive and non-competitive IC could be successfully used to monitor the HCV amplification performance, including the extraction, reverse transcription, amplification and detection steps, without compromising the detection of samples with low target concentrations. In conclusion, MS2-like particles generated by this strategy proved to be useful IC for RNA virus diagnosis, with advantage that they are produced by a low cost protocol. An attractive feature of this system is that it allows the construction of a multicontrol by the insertion of sequences from more than one pathogen, increasing its applicability for diagnosing different RNA viruses.

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“…As we had introduced NAT HCV 10,20 in 1998, our development was framed by the idea of within a similar methodological configuration, add HIV-RNA testing, at a minimal increase in cost and without affecting the sensitivity and overall performance of the test.…”

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confidence: 99%

Replacement of HIV p24 Ag test by a multiplex RT-PCR method for primary screening of blood donors

Levi

Wendel

Takaoka³

et al. 2007

Rev. Inst. Med. trop. S. Paulo

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SUMMARYNucleic Acid Testing (NAT) as a tool for primary screening of blood donors became a reality in the end of the 1990 decade. We report here the development of an "in-house" RT-PCR method that allows the simultaneous (multiplex) detection of HCV and HIV-RNA in addition to an artificial RNA employed as an external control. This method detects all HIV group M subtypes, plus group N and O, with a detection threshold of 500 IU/mL. After validation, the method replaced p24 Ag testing, in use for blood donation screening since 1996 at our services. From July 2001 to February 2006, 102,469 donations were tested and 41 (0.04%) were found HIV-RNA reactive. One NAT-only reactive donation (antibody non-reactive) was observed, with subsequent seroconversion of the implied donor, giving a yield of 1:102,469. This rate is in contrast to the international experience that reports a detection of approximately 1:600,000 -1:3,100,000 of isolated HIV-RNA donations.

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Primary screening of blood donors by nat testing for HCV-RNA: development of an "in-house" method and results

Cited by 16 publications

References 29 publications

A probable case of hepatitis B virus transfusion transmission revealed after a 13‐month‐long window period

A probable case of hepatitis B virus transfusion transmission revealed after a 13‐month‐long window period

Internal control for real-time polymerase chain reaction based on MS2 bacteriophage for RNA viruses diagnostics

Replacement of HIV p24 Ag test by a multiplex RT-PCR method for primary screening of blood donors

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