RESUMEN
The cystine/glutamate antiporter xCT is an important source of cysteine for cancer cells. Once taken up, cystine is reduced to cysteine and serves as a building block for the synthesis of glutathione, which efficiently protects cells from oxidative damage and prevents ferroptosis. As melanomas are particularly exposed to several sources of oxidative stress, we investigated the biological role of cysteine and glutathione supply by xCT in melanoma. xCT activity was abolished by genetic depletion in the Tyr::CreER; BrafCA; Ptenlox/+ melanoma model and by acute cystine withdrawal in melanoma cell lines. Both interventions profoundly impacted melanoma glutathione levels, but they were surprisingly well tolerated by murine melanomas in vivo and by most human melanoma cell lines in vitro. RNA sequencing of human melanoma cells revealed a strong adaptive upregulation of NRF2 and ATF4 pathways, which orchestrated the compensatory upregulation of genes involved in antioxidant defence and de novo cysteine biosynthesis. In addition, the joint activation of ATF4 and NRF2 triggered a phenotypic switch characterized by a reduction of differentiation genes and induction of pro-invasive features, which was also observed after erastin treatment or the inhibition of glutathione synthesis. NRF2 alone was capable of inducing the phenotypic switch in a transient manner. Together, our data show that cystine or glutathione levels regulate the phenotypic plasticity of melanoma cells by elevating ATF4 and NRF2.
Asunto(s)
Cisteína , Melanoma , Ratones , Animales , Humanos , Cisteína/metabolismo , Cistina , Compuestos de Sulfhidrilo , Melanoma/genética , Factor 2 Relacionado con NF-E2/genética , Factor 2 Relacionado con NF-E2/metabolismo , Glutatión/metabolismo , Estrés Oxidativo , Factor de Transcripción Activador 4/genética , Factor de Transcripción Activador 4/metabolismoRESUMEN
The redox regulator NRF2 becomes activated upon oxidative and electrophilic stress and orchestrates a response program associated with redox regulation, metabolism, tumor therapy resistance, and immune suppression. Here, we describe an unrecognized link between the integrated stress response (ISR) and NRF2 mediated by the ISR effector ATF4. The ISR is commonly activated after starvation or ER stress and plays a central role in tissue homeostasis and cancer plasticity. ATF4 increases NRF2 transcription and induces the glutathione-degrading enzyme CHAC1, which we now show to be critically important for maintaining NRF2 activation. In-depth analyses reveal that NRF2 supports ATF4-induced cells by increasing cystine uptake via the glutamate-cystine antiporter xCT. In addition, NRF2 upregulates genes mediating thioredoxin usage and regeneration, thus balancing the glutathione decrease. In conclusion, we demonstrate that the NRF2 response serves as second layer of the ISR, an observation highly relevant for the understanding of cellular resilience in health and disease.
Asunto(s)
Factor de Transcripción Activador 4 , Factor 2 Relacionado con NF-E2 , Neoplasias , Humanos , Factor de Transcripción Activador 4/genética , Factor de Transcripción Activador 4/metabolismo , Cistina/metabolismo , Glutatión/metabolismo , Factor 2 Relacionado con NF-E2/metabolismo , Estrés OxidativoRESUMEN
BACKGROUND: Viral safety of blood products in Germany has improved significantly over the last two decades. We describe the second documented transfusion-transmitted (TT) episode for the hepatitis C virus (HCV) in Germany since mandatory nucleic acid amplification techniques (NAT) screening was introduced in 1999. STUDY DESIGN AND METHODS: When a repeat donor who had tested negative for anti-HCV tested positive for HCV RNA by NAT in a minipool (MP) of eight, a look-back procedure was initiated. Qualitative, quantitative and genotyping assays were used to investigate the titers of the quarantined fresh frozen plasma (FFP) from the donor and a serum sample from the recipient of the pooled platelet concentrate (PPC). Amplified products of 5'UTR and HVR1 were used for sequence comparison to characterize the HCV genomic identity of donor and recipient samples. RESULTS: All NAT tests utilized in this procedure were able to detect a low HCV RNA titer (~15 IU/ml) in the FFP from the donation. Dilution of FFP by factor 8 was performed to mimic an MP, and the detection rate correlated well with the claimed sensitivity of the tests. Analysis of donor and recipient samples revealed genotype 3a viral transmission confirmed by sequence analysis. CONCLUSION: This TT HCV case could have been prevented by individual donation (ID) NAT. However, a low titer blood donation in the window period (WP) is very rare. Residual risk calculation for TT HCV in the WP revealed that, compared to MP-NAT testing, ID-NAT would improve blood safety only marginally.
Asunto(s)
Hepacivirus , Hepatitis C , Humanos , Hepacivirus/genética , Donantes de Sangre , Hepatitis C/diagnóstico , Alemania , ARN , Técnicas de Amplificación de Ácido Nucleico/métodos , Tamizaje MasivoRESUMEN
Infections with hepatitis B, C, and E virus (HBV, HCV, and HEV) can be transmitted via blood and cause severe acute or chronic liver infections. To ensure the safety of blood donations and protect recipients from virus transmissions, blood donations in Germany are tested for viral genomes using nucleic acid amplification techniques (NATs) as well as for viral antigens and antibodies by serological testing. This article describes the relevant regulations on the safety of blood and blood products in Germany and the various screening methods. The safety of blood products is assessed.Currently used NAT methods for detection of hepatitis viruses are based either on polymerase chain reaction (PCR) or isothermal methods such as transcription-mediated amplification (TMA), which enable a highly sensitive detection of viral infections and thereby contribute to the reduction of the diagnostic window. Antigen tests for the detection of viral surface protein of hepatitis B virus in blood donations were introduced in the 1970s in order to prevent potential transmissions. Since the introduction of mandatory testing for HCV-specific antibodies in 1992, HCV NAT testing in 1999, anti-HBc antibody testing in 2006, and the non-mandatory HBV NAT, which is voluntarily performed by most of the blood establishments, blood safety has increased tremendously. Only a few isolated cases of transfusion-transmitted infections in the early window period have been reported since. The success of the recent introduction of mandatory HEV NAT testing in 2020 will have to be assessed in the upcoming years. Besides blood donor screening, the system for blood safety in Germany is supplemented by additional measures for donor selection and pathogen inactivation.
Asunto(s)
Antígenos de Superficie de la Hepatitis B , Hepatitis B , Donantes de Sangre , Seguridad de la Sangre , ADN Viral , Alemania , Hepatitis B/diagnóstico , Hepatitis B/prevención & control , Humanos , Tamizaje MasivoRESUMEN
We report the sequences of two West Nile virus (WNV) strains (lineages 1 and 2) developed by the Paul-Ehrlich-Institut as reference materials. The materials are calibrated against the 1st World Health Organization WNV RNA International Standard and are intended for use in nucleic acid technology assays supporting transfusion safety.
RESUMEN
The European Directorate for the Quality of Medicines & HealthCare (EDQM) has run proficiency testing schemes on the detection of viral contaminants in human plasma pools by nucleic-acid amplification techniques since 1999 for hepatitis C virus and since 2004 for parvovirus B19. A retrospective analysis was performed to assess their impact and identify trends and progress in the results obtained by participating laboratories over a 15-year span, from 2004 to 2018. The results demonstrate that overall performance improved over that time, especially among the regular participants. Participation in these proficiency testing schemes is therefore recommended for all interested control laboratories. This analysis also shows that hepatitis C virus detection now seems well established compared to that of parvovirus B19, which still appears more challenging.
Asunto(s)
Hepacivirus/aislamiento & purificación , Parvovirus B19 Humano/aislamiento & purificación , Plasma/virología , Donantes de Sangre , ADN Viral/aislamiento & purificación , Hepacivirus/genética , Humanos , Parvovirus B19 Humano/genética , Estudios RetrospectivosRESUMEN
Receptor tyrosine kinases (RTK) are rarely mutated in cutaneous melanoma, but the expression and activation of several RTK family members are associated with a proinvasive phenotype and therapy resistance. Epidermal growth factor receptor (EGFR) is a member of the RTK family and is only expressed in a subgroup of melanomas with poor prognosis. The insight into regulators of EGFR expression and activation is important for the understanding of the development of this malignant melanoma phenotype. Here, we describe that the transcription factor NRF2, the master regulator of the oxidative and electrophilic stress response, mediates the expression and activation of EGFR in melanoma by elevating the levels of EGFR as well as its ligands EGF and TGFα. ChIP sequencing data show that NRF2 directly binds to the promoter of EGF, which contains a canonical antioxidant response element. Accordingly, EGF is induced by oxidative stress and is also increased in lung adenocarcinoma and head and neck carcinoma with mutationally activated NRF2. In contrast, regulation of EGFR and TGFA occurs by an indirect mechanism, which is enabled by the ability of NRF2 to block the activity of the melanocytic lineage factor MITF in melanoma. MITF effectively suppresses EGFR and TGFA expression and therefore serves as link between NRF2 and EGFR. As EGFR was previously described to stimulate NRF2 activity, the mutual activation of NRF2 and EGFR pathways was investigated. The presence of NRF2 was necessary for full EGFR pathway activation, as NRF2-knockout cells showed reduced AKT activation in response to EGF stimulation compared to controls. Conversely, EGF led to the nuclear localization and activation of NRF2, thereby demonstrating that NRF2 and EGFR are connected in a positive feedback loop in melanoma. In summary, our data show that the EGFR-positive melanoma phenotype is strongly supported by NRF2, thus revealing a novel maintenance mechanism for this clinically challenging melanoma subpopulation.
Asunto(s)
Melanoma/metabolismo , Factor 2 Relacionado con NF-E2/metabolismo , Transducción de Señal , Elementos de Respuesta Antioxidante , Secuencia de Bases , Sitios de Unión , Biomarcadores de Tumor , Proteínas Portadoras , Línea Celular Tumoral , Receptores ErbB/metabolismo , Humanos , Estimación de Kaplan-Meier , Ligandos , Melanoma/etiología , Melanoma/mortalidad , Melanoma/patología , Modelos Biológicos , Motivos de Nucleótidos , Unión ProteicaRESUMEN
The transcription factor NRF2 is the major mediator of oxidative stress responses and is closely connected to therapy resistance in tumors harboring activating mutations in the NRF2 pathway. In melanoma, such mutations are rare, and it is unclear to what extent melanomas rely on NRF2. Here we show that NRF2 suppresses the activity of the melanocyte lineage marker MITF in melanoma, thereby reducing the expression of pigmentation markers. Intriguingly, we furthermore identified NRF2 as key regulator of immune-modulating genes, linking oxidative stress with the induction of cyclooxygenase 2 (COX2) in an ATF4-dependent manner. COX2 is critical for the secretion of prostaglandin E2 and was strongly induced by H2O2 or TNFα only in presence of NRF2. Induction of MITF and depletion of COX2 and PGE2 were also observed in NRF2-deleted melanoma cells in vivo. Furthermore, genes corresponding to the innate immune response such as RSAD2 and IFIH1 were strongly elevated in absence of NRF2 and coincided with immune evasion parameters in human melanoma datasets. Even in vitro, NRF2 activation or prostaglandin E2 supplementation blunted the induction of the innate immune response in melanoma cells. Transcriptome analyses from lung adenocarcinomas indicate that the observed link between NRF2 and the innate immune response is not restricted to melanoma.
Asunto(s)
Ciclooxigenasa 2/metabolismo , Melanoma/patología , Factor 2 Relacionado con NF-E2/metabolismo , Neoplasias Cutáneas/patología , Factor de Transcripción Activador 4/metabolismo , Adenocarcinoma del Pulmón/genética , Adenocarcinoma del Pulmón/inmunología , Adenocarcinoma del Pulmón/patología , Animales , Diferenciación Celular/genética , Línea Celular Tumoral , Conjuntos de Datos como Asunto , Dinoprostona/metabolismo , Modelos Animales de Enfermedad , Femenino , Regulación Neoplásica de la Expresión Génica/inmunología , Técnicas de Silenciamiento del Gen , Técnicas de Inactivación de Genes , Humanos , Inmunidad Innata/genética , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/inmunología , Neoplasias Pulmonares/patología , Masculino , Melanoma/genética , Melanoma/inmunología , Ratones , Factor de Transcripción Asociado a Microftalmía/metabolismo , Factor 2 Relacionado con NF-E2/genética , Neoplasias Cutáneas/genética , Neoplasias Cutáneas/inmunología , Escape del Tumor/genéticaRESUMEN
BACKGROUND AND OBJECTIVES: Assessment of HBV-NAT testing compared to HBsAg and anti-HBc screening in German blood establishments for the period 2008-2015. MATERIALS AND METHODS: Blood donations screened for HBsAg and anti-HBc along with HBV-NAT were evaluated. Sensitivity of HBsAg and HBV-NAT tests was compared in 30 HBV seroconversion panels and with the viral load of the NAT-only cases. Residual risk for HBV in the WP was modelled. RESULTS: A total of 45 270 111 donations were evaluated. There were 29 NAT-only cases in the HBsAg-negative HBV-WP, one by ID-NAT and 28 by MP-NAT. MP-NAT, on average, showed higher sensitivity than HBsAg testing: MP-NAT-LoD of 146 IU/ml vs. 362 IU/ml HBV DNA for positive HBsAg detection (range 135-1502 IU/ml), resulting in 3·1 days (range 2·0-4·8 days) earlier HBV detection. Viral loads of the NAT-only cases confirmed the sensitivity of the HBV tests in the seroconversion study. One HBsAg-negative case was due to a new HBsAg mutant combination. There was one HBsAg-reactive only case. In addition, HBV incidence in the HBV-WP included 41 HBsAg-/HBV-NAT-positives and three HBV transmission cases. The residual risk for HBsAg was estimated to be 1:1 619 419-1 268 474 compared to 1:2 793 365-2 134 702 for MP-NAT. Within chronic HBV (HBsAg-/anti-HBc-positive and MP-NAT-negative) 70% were ID-NAT positive at low viral load (median 20 IU/ml). Among anti-HBc-only, supplementary ID-NAT detected 23 occult HBV infections. CONCLUSIONS: In the HBV-WP, MP-NAT provided a higher sensitivity than HBsAg testing, obtained a considerably higher yield and reduced the risk for HBV transmission. In later HBV stages, anti-HBc screening and HBV-ID-NAT intercepted potentially infectious donations.
Asunto(s)
Donantes de Sangre , Hepatitis B/epidemiología , Tamizaje Masivo/métodos , ADN Viral/sangre , Alemania/epidemiología , Hepatitis B/diagnóstico , Anticuerpos contra la Hepatitis B/sangre , Antígenos del Núcleo de la Hepatitis B , Antígenos de Superficie de la Hepatitis B/sangre , Virus de la Hepatitis B , Humanos , Sensibilidad y Especificidad , Carga ViralRESUMEN
Prolonged air leak is one of the most common postoperative complications in thoracic surgery. For elective partial lung resections, postoperative air leak lasting longer than 5â-â7 days is arbitrarily defined as prolonged. There are several predictive factors for the development of prolonged air leak that can be subdivided as either preoperative, such as obstructive pulmonary disease, or intraoperative risk factors, such as pleural adhesions or fused fissures. The treatment of postoperative prolonged air leak is performed on an individual basis for each patient and may vary considerably. Four major treatment options can be varied in sequence and use: intensified conservative therapy, installation of sclerosing agents or autologous blood, endoscopic procedures and surgical revision. Prevention, detection and treatment of postoperative prolonged air leak are common aspects of thoracic surgery with high clinical relevance. In contrast to standardised procedures in risk identification and diagnostic work-up, the treatment of prolonged air leak is performed on an individual basis and requires a great deal of surgical expertise. For effective targeted therapy, it seems advisable to agree on a specific standardised therapy sequence within a clinic.
Asunto(s)
Enfermedades Pleurales , Neumonectomía , Complicaciones Posoperatorias , Humanos , Factores de RiesgoRESUMEN
BACKGROUND: RNA viruses are associated with a high frequency of mutations because of the missing proofreading function of polymerases, such as reverse transcriptase. Between 2007 and 2010, six blood donations with false-negative nucleic acid technology (NAT) results were reported in Germany. Therefore, NAT screening in two viral genome regions was introduced by our blood donation service in 2010 on a voluntary basis and became mandatory in Germany since the beginning of 2015. STUDY DESIGN AND METHODS: Blood donor screening was done using, in parallel, the German Red Cross (GRC) HIV-1 CE long terminate repeats (LTR) PCR kit and the GRC HIV-1 gag CE PCR kit. In total, 7 million blood donations were screened during the study period from 2010 to 2014 with the GRC dual-target human immunodeficiency virus 1 (HIV-1) NAT system. Additionally, three suspicious specimens were analyzed by four monotargeted NAT assays and by five dual-target NAT assays. RESULTS: Three of 7 million donations tested negative using the 5'LTR-polymerase chain reaction, but they were positive if amplification was performed in the gag region. HIV antibodies were detected in all three donations. Nucleic acid sequence analysis identified a deletion of 22 bases within the 5'LTR probe binding region. Three different ltr-based monotargeted assays missed two donations, except for a low-reactive result obtained by one of the assays. In total, the detection rates for HIV-1-positive donations were 37.5% (3/8) for monotargeted assays and 100% (10/10) for dual-target assays. CONCLUSION: The current data demonstrate that dual-target NAT systems reduce the risk of false-negative HIV-1 NAT screening results.
Asunto(s)
Donantes de Sangre , Duplicado del Terminal Largo de VIH , VIH-1 , ARN Viral , Juego de Reactivos para Diagnóstico , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Productos del Gen gag del Virus de la Inmunodeficiencia Humana , Seguridad de la Sangre , Selección de Donante , Femenino , Alemania , VIH-1/genética , VIH-1/metabolismo , Humanos , Masculino , ARN Viral/sangre , ARN Viral/genética , Cruz Roja , Estudios Retrospectivos , Productos del Gen gag del Virus de la Inmunodeficiencia Humana/sangre , Productos del Gen gag del Virus de la Inmunodeficiencia Humana/genéticaRESUMEN
Between 1 June and 31 December 2016, 13,023 blood donations from the University Hospital Aachen in Germany were routinely screened for West Nile virus (WNV) RNA using the cobas TaqScreen WNV Test. On 28 September 2016, one blood donor was tested positive. Subsequent analysis revealed an acute Usutu virus (USUV) infection. During the ongoing USUV epizootics in Germany, blood transfusion services, public health authorities and clinicians should be aware of increased human USUV infections.
Asunto(s)
Infecciones por Flavivirus/diagnóstico , Fiebre del Nilo Occidental/diagnóstico , Adulto , Anticuerpos Antivirales/inmunología , Donantes de Sangre , Virus de la Encefalitis Japonesa (Especie)/inmunología , Virus de la Encefalitis Transmitidos por Garrapatas/inmunología , Femenino , Flavivirus/genética , Flavivirus/inmunología , Infecciones por Flavivirus/inmunología , Alemania/epidemiología , Humanos , Inmunoglobulina G/inmunología , Inmunoglobulina M/inmunología , Tamizaje Masivo , ARN Viral/sangre , ARN Viral/genética , Fiebre del Nilo Occidental/inmunología , Virus del Nilo Occidental/genética , Virus del Nilo Occidental/inmunologíaRESUMEN
Presenilin 1 (PSEN1) mutations are the major cause of autosomal dominant Alzheimer's disease (ADAD). Here we report three novel PSEN1 mutations: Ile238_Lys239insIle, Ala246Pro and Ala164Val from patients who manifested in their twenties, forties and seventies, respectively, with variant clinical presentations of dementia. These cases exemplify the tremendous heterogeneity of clinical phenotypes and age of onset associated with PSEN1 mutations. The possibility of ADAD--not previously suspected in two of our patients--should always be considered in neurodegenerative conditions albeit they might neither exhibit the typical clinical picture of Alzheimer's disease nor early onset dementia, which is regarded the primary clinical sign of hereditary neurodegeneration.
Asunto(s)
Enfermedad de Alzheimer/genética , Mutación , Presenilina-1/genética , Adulto , Edad de Inicio , Anciano , Enfermedad de Alzheimer/fisiopatología , Resultado Fatal , Femenino , Humanos , Masculino , Persona de Mediana Edad , FenotipoRESUMEN
Nucleic acid amplification technique (NAT)-based assays (referred to here as NAT assays) are increasingly used as an alternative to culture-based approaches for the detection of mycoplasma contamination of cell cultures. Assay features, like the limit of detection or quantification, vary widely between different mycoplasma NAT assays. Biological reference materials may be useful for harmonization of mycoplasma NAT assays. An international feasibility study included lyophilized preparations of four distantly related mycoplasma species (Acholeplasma laidlawii, Mycoplasma fermentans, M. orale, M. pneumoniae) at different concentrations which were analyzed by 21 laboratories using 26 NAT assays with a qualitative, semiquantitative, or quantitative design. An M. fermentans preparation was shown to decrease the interassay variation when used as a common reference material. The preparation was remanufactured and characterized in a comparability study, and its potency (in NAT-detectable units) across different NATs was determined. The World Health Organization (WHO) Expert Committee on Biological Standardization (ECBS) established this preparation to be the "1st World Health Organization international standard for mycoplasma DNA for nucleic acid amplification technique-based assays designed for generic mycoplasma detection" (WHO Tech Rep Ser 987:42, 2014) with a potency of 200,000 IU/ml. This WHO international standard is now available as a reference preparation for characterization of NAT assays, e.g., for determination of analytic sensitivity, for calibration of quantitative assays in a common unitage, and for defining regulatory requirements in the field of mycoplasma testing.
Asunto(s)
ADN Bacteriano/genética , Mycoplasma/genética , Técnicas de Amplificación de Ácido Nucleico/normas , Laboratorios/normas , Mycoplasma/clasificación , Mycoplasma/aislamiento & purificación , Técnicas de Amplificación de Ácido Nucleico/métodos , Organización Mundial de la SaludRESUMEN
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 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.
RESUMEN
BACKGROUND: Nucleic acid amplification techniques (NAT) in routine blood donor screening considerably reduce the diagnostic window phase period. Nevertheless, several reports of false-negative NAT results were published. Here, four cases of human immunodeficiency virus Type 1 (HIV-1) RNA-positive blood donations that escaped detection by NAT screening are described. STUDY DESIGN AND METHODS: A total of 2.7 million blood donations were screened for viral infections between January 2010 and October 2012 in our German Red Cross blood donation service. Four plasma specimens with false-negative NAT results were comparatively investigated with 12 CE-marked NAT assays. In two cases of putative HIV-1 variants the target region of the NAT assay was sequenced allowing comparison with the respective primers and probes. RESULTS: Most of the NAT assays used in routine blood donor screening with the 5'-long terminal repeat (LTR) as target region demonstrated deficiencies in detecting the viral variants and the low-viral-carrier donations. Sequence analysis revealed in one case a deletion of 56 nucleotides within the 5'-LTR preventing the binding of the probe accompanied by a neighbored insertion of another 52 nucleotides and several primer mismatches in another case. No false-negative results were obtained for these cases using dual-target assays. The viral load of the remaining two false-negative results was below the NAT's limit of detection. CONCLUSION: HIV-1 is characterized by a high mutation rate and rapid generation of new viral variants. By the use of one target region for HIV-1 NAT assays there is a certain risk of false-negative results. Employing HIV-1 multi- and dual-target assays in routine blood donor screening seems to be a reasonable possibility to minimize this problem.
Asunto(s)
Seguridad de la Sangre/métodos , Errores Diagnósticos , Infecciones por VIH/diagnóstico , VIH-1/aislamiento & purificación , Técnicas de Amplificación de Ácido Nucleico , Adolescente , Adulto , Secuencia de Bases , Donantes de Sangre/estadística & datos numéricos , Seguridad de la Sangre/normas , Seguridad de la Sangre/estadística & datos numéricos , Reacciones Falso Negativas , Femenino , Variación Genética , Infecciones por VIH/sangre , Infecciones por VIH/epidemiología , Infecciones por VIH/transmisión , VIH-1/genética , Humanos , Masculino , Tamizaje Masivo/métodos , Tamizaje Masivo/normas , Tamizaje Masivo/estadística & datos numéricos , Persona de Mediana Edad , Datos de Secuencia Molecular , Pruebas Serológicas/métodos , Pruebas Serológicas/normas , Pruebas Serológicas/estadística & datos numéricosRESUMEN
BACKGROUND: Five cases of human immunodeficiency virus Type 1 (HIV-1) RNA-positive blood donations are described that escaped detection by three different CE-marked nucleic acid amplification technique (NAT) screening assays. These events were associated with two HIV-1 transmissions to recipients of blood components. The implicated NAT assays are monotarget assays and amplify in different viral genome regions (group-specific antigen or long terminal repeat). Investigations into the cause of the false-negative test results were initiated. STUDY DESIGN AND METHODS: Plasma specimens of the five NAT false-negative cases were comparatively investigated in 12 CE-marked HIV-1 NAT systems of differing design. The relative amplification efficiency for the HIV-1 variant was determined for each assay. Sequencing of the variants in the region targeted by each false-negative NAT assay allowed comparison with the respective primers and probes. RESULTS: Some of the NAT assays designed in a similar way to false-negative monotarget NATs also revealed deficiencies in detecting the viral variants. In each case sequencing of the assay target region in the variants demonstrated mismatches with primers and probes used by the assays. Some dual-target assays showed decreased amplification efficiency, but not false-negative results. CONCLUSION: HIV is characterized by its rapid evolution of new viral variants. The evolution of new sequences is unpredictable; NAT screening assays with a single target region appear to be more vulnerable to sequence variations than dual-target assays. Based on this experience with false-negative tests results by monotarget NAT assays, the Paul-Ehrlich-Institut is considering requesting dual-target NAT assays for HIV-1 blood donation screening in Germany.
Asunto(s)
Sondas de ADN/fisiología , Infecciones por VIH/diagnóstico , VIH-1/genética , Tamizaje Masivo/métodos , Técnicas de Amplificación de Ácido Nucleico , Adolescente , Adulto , Donantes de Sangre , Sondas de ADN/química , Sondas de ADN/genética , Reacciones Falso Negativas , Infecciones por VIH/sangre , Infecciones por VIH/prevención & control , Infecciones por VIH/virología , Seropositividad para VIH/sangre , Seropositividad para VIH/genética , Seropositividad para VIH/transmisión , VIH-1/aislamiento & purificación , Humanos , Masculino , Tamizaje Masivo/normas , Técnicas de Amplificación de Ácido Nucleico/métodos , Técnicas de Amplificación de Ácido Nucleico/normas , ARN Viral/sangre , ARN Viral/genética , Carga ViralRESUMEN
BACKGROUND: Mandatory nucleic acid test (NAT) blood screening was introduced in Germany in 1999 for hepatitis C virus (HCV) RNA and in 2004 for human immunodeficiency virus Type 1 (HIV-1) RNA. Minimal sensitivity limits of 5000 IU HCV RNA/mL and 10,000 IU HIV-1 RNA/mL were defined for the individual donation facilitating testing of minipools (MPs). The NAT yield obtained from all blood organizations is summarized. Transfusion-associated virus transmissions despite NAT screening ("breakthrough transmissions") are analyzed. STUDY DESIGN AND METHODS: In Germany, a variety of NAT assays is applied for NAT screening pool sizes of up to 96 donations. Subsets of NAT yield cases were characterized with regard to viral loads by quantitative NAT and with regard to viral genotypes. Confirmed breakthrough transmissions were analyzed using different molecular and serologic assays. RESULTS: Ninety-two HCV NAT yield cases among 40.8 million and 11 HIV-1 NAT yield cases among 17.1 million donations were identified. During this period, one transmission case was confirmed for HCV and one for HIV-1. The two incidents escaped NAT detection because of low-level viremia and/or suboptimal amplification efficiency. Evidence was obtained for a case of HIV-1 nontransmission by a low-level HIV-1 contaminated red blood cell unit. CONCLUSION: NAT screening of MPs identified the vast majority of window-phase donations. A significant number of transmission cases was interdicted; breakthrough transmissions may still occur as rare events, even with individual-donation NAT in place. Sensitivity limits might be adapted to the current "state of the art" taking account of viral dynamics during early infection, incidence rates, and costs.