RESUMO
BACKGROUND AND OBJECTIVES: Two External Quality Assessment Programmes (EQAPs) were run in 2008 and 2009 to evaluate the proficiency of blood centres in detecting, by nucleic acid amplification techniques (NAT), the possible contamination of plasma with hepatitis C virus (HCV), human immunodeficiency virus (HIV) and hepatitis B virus (HBV). MATERIALS AND METHODS: In the EQAP-2008, three customized panels were designed; each containing positive samples with a viral nominal concentration for the three viruses of about three times the 95% DL of the respective commercial NAT assay. In the EQAP-2009, the proficiency of the participants was evaluated with a single panel, independently on the NAT method used. RESULTS: While 84% (102/122) of the participants in the EQAP-2008 correctly identified the positive and negative samples of the panels, in the EQAP-2009 the percentage of proficient laboratories increased to 97% (118/122). Most importantly, in this 2-year experience, we observed a decrease in the number of pre-/postanalytical errors, from 14 in 2008 to two in 2009. CONCLUSIONS: The design of these two EQAPs allowed participants to assess the performance of the NAT methods applied in their routine screening of blood donations, not only with respect to analytical errors but also to human errors that, despite the high level of automation reached by NAT methods, can still occur.
Assuntos
Bancos de Sangue , DNA Viral/sangue , HIV , Hepacivirus , Vírus da Hepatite B , Técnicas de Amplificação de Ácido Nucleico/normas , Garantia da Qualidade dos Cuidados de Saúde , RNA Viral/sangue , Feminino , Humanos , Itália , Masculino , Técnicas de Amplificação de Ácido Nucleico/métodos , Sensibilidade e EspecificidadeRESUMO
BACKGROUND AND OBJECTIVES: In this EQA study a novel approach was used to assess the performance of blood centres and blood product manufacturers in detecting the possible contamination of plasma with HCV, HIV and HBV by NAT. MATERIALS AND METHODS: A panel of 12 samples, three negative and three positive for each virus, was distributed to the EQA participants. The positive samples were prepared, using the respective WHO standards, in order to obtain a viral concentration of about three times the 95% DL of the methods most commonly used by laboratories involved in blood screening by NAT. Participants were requested to test each sample of the panel on different days, possibly by different operators using their routine NAT assay. RESULTS: Overall, the participants' performance was satisfactory. In particular, 49 of the 59 participants (83%) were able to correctly identify all samples. Regarding the remaining 10 laboratories, in three cases a deviation from the laboratory's procedure that could be attributed to an operator's mistake was observed, in two cases a possible cross-contamination occurred while in the remaining five cases the failure to detect the positive samples couldn't be ascribed to any relevant deviation in the laboratory's procedure. CONCLUSIONS: The novel design of this EQA study allowed participants to verify their day by day activity as the study was carried out in the context of their routine testing. Under these conditions, it was demonstrated that, despite the high level of automation reached by NAT assays, human errors can still occur.
Assuntos
HIV/isolamento & purificação , Hepacivirus/isolamento & purificação , Vírus da Hepatite B/isolamento & purificação , Técnicas de Amplificação de Ácido Nucleico/métodos , Controle de Qualidade , DNA Viral/sangue , HIV/genética , Hepacivirus/genética , Vírus da Hepatite B/genética , Humanos , Variações Dependentes do Observador , RNA Viral/sangueRESUMO
BACKGROUND AND OBJECTIVES: This External Quality Assessment (EQA) study was aimed at assessing the proficiency of blood centres and blood product manufacturers in detecting, by nucleic acid amplification technology (NAT), the possible contamination of plasma with hepatitis C virus (HCV), human immunodeficiency virus (HIV) and hepatitis B virus (HBV). MATERIALS AND METHODS: Three independent panels, one for each virus, were prepared at the Istituto Superiore di Sanità (ISS) by diluting the respective reference preparations. NAT methods used by the EQA participants included polymerase chain reaction (PCR) assays by Roche, transcription-mediated amplification (TMA) assays by Chiron and in-house PCR assays. RESULTS: Forty-three of the 45 participants (95.6%) in the HCV EQA/5 who used a validated method were consistently able to detect a nominal concentration of 100 IU/ml for all six major genotypes. In the case of the HIV EQA/1, all 35 participants detected the samples containing 1000 IU/ml HIV, while five (14.3%) did not identify the samples containing 100 IU/ml HIV. With respect to the HBV EQA/1, all 16 participants correctly identified the positive samples containing either 1000 IU/ml or 100 IU/ml HBV. No false-positive results were observed with any of the three panels. CONCLUSIONS: The HCV EQA/5 showed an improved proficiency of laboratories as compared with the HCV EQA/4. In fact, HCV genotypes 1, 2, 3 and 5 were correctly identified in 100% of the assays and genotypes 4 and 6 in 97.8% of the assays. While most of the participants in the HIV EQA/1 showed a good level of proficiency, an excellent performance was shown by all participants in the HBV EQA/1.
Assuntos
HIV/isolamento & purificação , Hepacivirus/isolamento & purificação , Vírus da Hepatite B/isolamento & purificação , Técnicas de Amplificação de Ácido Nucleico/normas , Plasma/virologia , Reações Falso-Positivas , Humanos , Reação em Cadeia da Polimerase/normas , Garantia da Qualidade dos Cuidados de SaúdeAssuntos
Doadores de Sangue , Infecções por Herpesviridae/epidemiologia , Herpesvirus Humano 8 , Hospedeiro Imunocomprometido , Imunoglobulinas Intravenosas , Infecções por Herpesviridae/etiologia , Infecções por Herpesviridae/imunologia , Humanos , Imunoglobulinas Intravenosas/efeitos adversos , Itália , Fatores de RiscoAssuntos
Anticorpos Anti-Hepatite/sangue , Antígenos de Hepatite/imunologia , Hepatite Viral Humana/prevenção & controle , Imunoglobulinas Intravenosas/imunologia , Imunoglobulinas/imunologia , Glicoproteínas de Membrana/imunologia , Proteínas do Envelope Viral/imunologia , Contaminação de Medicamentos , HumanosRESUMO
We have used the polymerase chain reaction (PCR) to detect by co-amplification, multiple regions of the HIV-1 genome in infected cells. Genomic RNA and DNA from productively infected H9 cells were independently extracted and amplified in reactions with and without reverse transcriptase respectively using primer pairs to the gag, env, tat and nef regions of the viral genome in the same reaction mixture. PCR-products were analysed by liquid hybridization with end labelled oligonucleotide probes followed by gel-electrophoresis (oligomer hybridization). The primer pairs were capable of detecting as few as 10 copies of RNA and 10-20 copies of integrated proviral DNA. The ability to co-amplify multiple target regions in the same incubation mixture provides a method for detecting and confirming the presence of HIV-1 in samples for which limited nucleic acid is available. In addition, in reconstitution experiments, the same method was used to detect HIV-1 and HTLV-I simultaneously with comparable sensitivity (20-40 gene copies each). This offers the possibility of simultaneous diagnosis of multiple viral infections, such as those that occur in AIDS, on the same sample preparation.