RESUMO
AIMS: To evaluate the potential use of synthetic oligonucleotides as a standard curve for proviral load (PVL) of human T-cell leukaemia virus type 1 (HTLV-1) quantification in peripheral blood mononuclear cells (PBMC) of HTLV-1-infected individuals by quantitative real-time polymerase chain reaction (qPCR) analysis. METHODS AND RESULTS: Synthetic oligonucleotides based on HTLV-1 genome were customized to use as a standard curve. Twelve anti-HTLV-1-positive samples with known HTLV-1 PVL, previously quantified by qPCR assay using TARL-2 cells as a conventional standard curve, were submitted to the new protocol. The proviral quantification levels had a high concordance with qPCR results using a conventional standard curve. The results demonstrate that the conventional standard curve can be replaced by a synthetic standard curve due to its ability to quantification based on the linearity and qPCR efficiency and similar results with a validated qPCR assay using a conventional standard curve. CONCLUSIONS: Synthetic oligonucleotides standard curves could be a very useful tool on HTLV-1 diagnosis and absolute HTLV-1 PVL quantification. SIGNIFICANCE AND IMPACT OF THE STUDY: HTLV-1 PVL determination using synthetic oligonucleotides standard curve by qPCR could be a helpful alternative for the laboratories that monitor infected patients as an important prognostic factor in HTLV-1-associated diseases progression. Also, it can decrease costs and overcome the biological limitations of the plasmid curve.
Assuntos
Infecções por HTLV-I/diagnóstico , Vírus Linfotrópico T Tipo 1 Humano/isolamento & purificação , Provírus/isolamento & purificação , Reação em Cadeia da Polimerase em Tempo Real/métodos , Carga Viral/métodos , Adulto , DNA Viral/genética , Progressão da Doença , Genoma Viral/genética , Infecções por HTLV-I/sangue , Infecções por HTLV-I/virologia , Vírus Linfotrópico T Tipo 1 Humano/genética , Humanos , Leucócitos Mononucleares/virologia , Pessoa de Meia-Idade , Oligonucleotídeos/síntese química , Oligonucleotídeos/genética , Prognóstico , Provírus/genética , Reação em Cadeia da Polimerase em Tempo Real/normas , Carga Viral/normasRESUMO
Foot-and-mouth-disease (FMD) is a highly contagious disease of domestic animals which can result in substantial economic losses, caused by the FMD virus (FMDV). The aim of this study was to develop and standardize a novel reverse transcriptase droplet digital PCR (RT-ddPCR) assay for the quantification of FMDV RNA. This assay was based upon an OIE-recognized real-time RT-PCR that detects the 3D-encoding region of FMDV. The limit of detection at 101.4 TCID50/mL and 26.5 copies was determined using FMDV-A24-Cruzeiro-virus and a plasmid containing the 3D-FMDV sequences, respectively. FMDV O, A and C serotypes and 11 species of non-FMDV were used to confirm the sensitivity and specificity of the assay. The RT-ddPCR was standardized using 60 bovine samples (representing negative and positive samples of epithelium and/or oesophageal-pharyngeal [OP] fluid) from animals suspected of vesicular diseases and previously tested by RT-qPCR. The RT-ddPCR showed robustness, sensitivity, specificity and accuracy, with similar results to the RT-qPCR. Moreover, the new RT-ddPCR diagnostic tool allowed the absolute quantification of FMDV RNA from epithelium and OP-fluid samples, as well as having the advantages of direct quantification by endpoint, eliminating the need for a calibration standard curve required in quantitative real-time RT-PCR.
Assuntos
Vírus da Febre Aftosa/isolamento & purificação , Febre Aftosa/virologia , RNA Viral/análise , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Carga Viral/métodos , Animais , Bovinos , Doenças dos Bovinos/virologia , Vírus da Febre Aftosa/genética , RNA Viral/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa/normas , Sensibilidade e Especificidade , Sorogrupo , Carga Viral/normasRESUMO
Quantitative real-time PCR (qPCR) is increasingly being used for the detection of bovine leukemia virus (BLV) proviral DNA. Nevertheless, quality control for the validation and standardization of such tests is currently lacking. Therefore, the present study was initiated by three Office International des Epizooties (OIE) reference laboratories and three collaborating laboratories to measure the interlaboratory variability of six already developed and available BLV qPCR assays. For that purpose, an international panel of 58 DNA samples reflecting the dynamic range of the majority of the assays was distributed to six testing centers. Based on qualitative results, the overall agreement among all six laboratories was moderate. However, significant variability in the measurement of the BLV proviral DNA copy number was observed among different laboratories. Quantitative PCR assays, even when performed by experienced staff, can yield large variability in BLV proviral DNA copy numbers without harmonization. Further standardization of different factors (i.e., utilization of unified protocols and unique calibrators) should increase interlaboratory agreement.
Assuntos
Leucose Enzoótica Bovina/diagnóstico , Vírus da Leucemia Bovina/fisiologia , Provírus/genética , Reação em Cadeia da Polimerase em Tempo Real/normas , Carga Viral/métodos , Animais , Bovinos , Testes Diagnósticos de Rotina/normas , Laboratórios/normas , Vírus da Leucemia Bovina/genética , RNA Viral/genética , Carga Viral/normasRESUMO
A quantitative real-time PCR (qPCR) assay using SYBR Green dye was established in order to detect and quantify the proviral DNA of HTLV-1 in peripheral blood mononuclear cells (PBMCs). Primers were designed, and the assay was standardized to amplify a novel, conserved HTLV-1 tax region. Proviral load was normalized to the amount of cellular DNA by quantitation of the human albumin gene. Firstly, the qPCR was assessed determining the specificity, sensitivity, dynamic range and intra- and inter-assay reproducibility of the technique. The limit of detection as determined by PROBIT analysis using dilutions of the standard was 2.97 copies. The assay had an excellent dynamic range from 105 to 10¹ copies per reaction and good intra- and inter-assay reproducibility, CVs less than 2%. Secondly, the performance of the qPCR was tested on 40 HTLV-1 seropositive individuals. Proviral load for HTLV-1 carriers ranged from 2.2×10² to more than 8.3×104 copies/106 PBMCs. The high sensitivity and wide dynamic range allowed the determination of a broad range of HTLV-1 proviral loads in infected individuals. This assay is a valuable alternative diagnostic tool when current available serological assays are insufficient. In addition, it will facilitate the study of the relationship between proviral load and pathogenesis.
Assuntos
Genes pX , Infecções por HTLV-I/diagnóstico , Infecções por HTLV-I/virologia , Vírus Linfotrópico T Tipo 1 Humano/isolamento & purificação , Provírus/isolamento & purificação , Reação em Cadeia da Polimerase em Tempo Real/métodos , Carga Viral/métodos , Primers do DNA/genética , Vírus Linfotrópico T Tipo 1 Humano/genética , Humanos , Provírus/genética , Reação em Cadeia da Polimerase em Tempo Real/normas , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Carga Viral/normasRESUMO
We examined failures of commercial human immunodeficiency virus type 1 (HIV-1) viral load assays of 1,195 plasma samples from Brazilian patients. Assay failure was assumed for samples in which the virus was undetectable by commercial assay but which tested positive by real-time reverse transcription-PCR of the HIV-1 long terminal repeat (LTR) region or if the viral load differed by >2 log10 from that determined by LTR assay. Failure rates for Bayer Versant bDNA 3.0, Roche Amplicor Monitor v1.5, and bioMerieux NucliSens QT were 0.68, 0.47, and 4.33%, respectively. NucliSens may be inadequate for use in Brazil.
Assuntos
Erros de Diagnóstico , Infecções por HIV/virologia , HIV-1/fisiologia , RNA Viral/sangue , Kit de Reagentes para Diagnóstico , Carga Viral , Sequência de Bases , Ensaio de Amplificação de Sinal de DNA Ramificado , Brasil , Repetição Terminal Longa de HIV/genética , HIV-1/classificação , HIV-1/genética , HIV-1/isolamento & purificação , Humanos , Dados de Sequência Molecular , Técnicas de Amplificação de Ácido Nucleico , RNA Viral/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Análise de Sequência de DNA , Carga Viral/normasRESUMO
La medición de la cantidad de virus de la inmunodeficiencia humana es el pricipal criterio de laboratorio en el manejo de la terapia con antirretrovirales. Uno de los objetivos más importantes del tratamiento es buscar el medicamento o la combinación de éstos que logre reducir el número de partículas o copias virales con el fin de dismunuir el efecto de la infección sobre el sistema inmunológico. La cantidad de virus circulante, conocida como carga viral, aparte de determinar el estado de la infección, es el mejor criterio para evaluar la respuesta al tratamiento. También es un importante marcador de pronóstico y progresión de la enfermedad cuando se utiliza apropiadamente y en conjunción con otras pruebas, como la determinación de las subpoblaciones de linfocitos T, provee al médico una invaluable herramienta para el tratamiento individual del paciente por virus de la inmunodeficiencia humana. Se presenta una revisión completa de la carga viral como prueba de laboratorio y se propone su utilización en el tratamiento de los pacientes infectados con virus de la inmunodeficiencia humana; todo acorde con la literatura disponible y las recomendaciones del Ministerio de Salud de colombia.
Assuntos
Humanos , Carga Viral/normas , Carga Viral/tendências , Carga Viral/estatística & dados numéricos , Síndrome da Imunodeficiência Adquirida/diagnóstico , Síndrome da Imunodeficiência Adquirida/fisiopatologiaRESUMO
Para las infecciones causadas por agentes virales como el virus de la inmunodeficiencia humana (VIH), las pruebas más utilizadas se basan en la determinación de anticuerpos especificos producidos por el sistema inmune del paciente como respuesta al agente infeccioso. Estas pruebas que determinan anticuerpos tienen utilidad para estudios de tamización y como herramienta diagnóstica, pero sólo permiten una medida indirecta de la infección viral. De otra parte, el recuento de linfocitos CD4+, que se utiliza para la profilaxis de infecciones oportunistas y seguimiento de los pacientes con VIH, ha demostrado ser poco efectivo para la evaluación de la respuesta al tratamiento. El desarrollo de nuevas técnicas moleculares como la prueba de ADN- ramificado -bDNA- para la cuantificaión del ARN del VIH-1 -carga viral- en plasma, finalmente ha permitido el estudio individualizado de la progresión clínica de la enfermedad y de la respuesta a la terapia antiviral. Se describe paso a paso el proseso de esta técnica, al igual que sus ventajas y desventajas ante a las otras pruebas utilizadas para la evaluación de los pacientes infectados por el VIH.