RESUMO
In order to characterize A/H5N1 viral sequences, a bioinformatics approach accurately identified viral sequences from discovery of a sequence signature, which provided enough distinctive information for sequence identification. Eight highly pathogenic H5N1 viral isolations were collected from different areas of Thailand between 2003 and 2006, and were used for analysis of H5N1 genotypic testing with a semiconductor-based oligonucleotide microarray. All H5N1 samples and H1N1, H4N8 negative controls were correctly subtyped. Sensitivity of the eight oligonucleotide probes, with optimized cut-offs, ranged from 70% (95% CI 65-75) to 87% (95% CI 84-91), and the corresponding Kappa values ranged from 0.76 (95% CI 0.72-0.80) to 0.86 (95% CI 0.83-0.89). Semi-conductor-based oligonucleotide array and oligonucleotide probes corresponded well when detecting H5N1. After fully correcting the subtype from the result of microarray signal intensity, the microarray output method combined with bioinformatics tools, identified and monitored genetic variations of H5N1. Capability of distinguishing different strains of H5N1 from Thailand was the outstanding feature of this assay. Ninety percent of HA and NA (4/5) genes were sequenced correctly, in accordance with previous examinations performed by classical diagnostic methods. The low-medium-high bioinformatics resolutions were able to predict an epidemic strain of H5N1. This study also showed the advantage of using a large genotypic database to predict the epidemic strain of H5N1. However, the monitoring protocol of this new strain has been recommended for further study with a large-scale sample.
Assuntos
Biologia Computacional/métodos , DNA Viral/genética , Monitoramento Ambiental/métodos , Virus da Influenza A Subtipo H5N1/classificação , Virus da Influenza A Subtipo H5N1/genética , Vacinas contra Influenza/síntese química , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Animais , Sequência de Bases , Sequência Consenso , Sondas de DNA/metabolismo , Eletroforese em Gel de Ágar , Virus da Influenza A Subtipo H5N1/isolamento & purificação , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Sensibilidade e Especificidade , Homologia de Sequência do Ácido Nucleico , TailândiaRESUMO
BACKGROUND: A fixed-dose combination of stavudine, lamivudine, and nevirapine (d4T/3TC/NVP) is extensively used as initial antiretroviral regimen in developing countries. K65R mutations that occur after failing this regimen prevent the use of tenofovir, didanosine, and abcavir in the second-line regimen. OBJECTIVES: To determine the prevalence and risk factors of K65R mutations after failing d4T/3TC/NVP. STUDY DESIGN: Genotypic resistance testing was conducted among HIV-1 infected patients who experienced virological failure with an initial regimen of d4T/3TC/NVP. RESULTS: There were 122 patients who received antiretroviral therapy (ART) for a median (IQR) duration of 19 (13-27) months. Median (IQR) CD4 cell count and plasma HIV-1 RNA at virological failure was 174 (109-264) cells/mm(3) and 4.0 (3.7-4.6)log copies/mL, respectively. The prevalence of K65R mutations was 7%. Patients with K65R mutations had higher plasma HIV-1 RNA at virological failure compared to patients without K65R mutations (4.9log copies/mL vs. 4.0log copies/mL, p=0.001). By logistic regression analysis only plasma HIV-1 RNA at failure correlated with the occurrence of K65R mutations [OR 4.2 (95% CI, 1.5-11.2) per 0.5log copies/mL increment of HIV-1 RNA]. CONCLUSIONS: Seven percent of patients had K65R mutations after failing an initial d4T/3TC/NVP regimen. Tenofovir, didanosine, and abcavir cannot be used in second-line regimen for these patients. HIV-1 RNA at the time that virological failure was detected correlated with the occurrence of K65R mutations.
Assuntos
Substituição de Aminoácidos/genética , Fármacos Anti-HIV/uso terapêutico , Farmacorresistência Viral/genética , Infecções por HIV/virologia , HIV-1/genética , Lamivudina/uso terapêutico , Nevirapina/uso terapêutico , Estavudina/uso terapêutico , Adulto , Contagem de Linfócito CD4 , Feminino , Infecções por HIV/tratamento farmacológico , Infecções por HIV/imunologia , HIV-1/isolamento & purificação , Humanos , Masculino , Mutação de Sentido Incorreto , Prevalência , RNA Viral/sangue , Fatores de Risco , Carga ViralRESUMO
HIV-1 viral load is a basic marker to evaluate the severity of HIV-1 related diseases and to monitor the effectiveness of treatment. A method based on real-time RT-PCR technology has been developed to quantify HIV-1 RNA using self-quenched fluorogenic primers known as LUX primers. They were used in this study to recognize a low variable gag region of subtype E and B consensus sequences. Specificity was verified by amplicon melting temperatures. An external standard curve was constructed with 10 fold serial dilutions of synthetic HIV-gag RNA. A broad range linear relationship (10 to 10(6) copies/ml) was observed between the number of PCR cycles needed to detect a fluorescent signal and the number of RNA copies. The intra- and inter-assay coefficients of variation were 0.72 to 2.54% and 3.14 to 8.83%, respectively, thus indicating good reproducibility. Thirty out of fifty HIV-infected individual plasma samples were quantified by this method and compared with the AMPLICOR HIV-1 Monitor assay, which is widely considered the reference technique for HIV-RNA viral load measurement. The results indicate that the AMPLICOR HIV-1 Monitor assay and real-time RT-PCR using LUX primers are in good agreement (mean difference in log10 copies/ml+/-2 standard deviations = 0.21+/-1.34).
Assuntos
Infecções por HIV/sangue , HIV-1 , RNA Viral/sangue , Carga Viral , Humanos , Padrões de Referência , Reação em Cadeia da Polimerase Via Transcriptase Reversa , TailândiaRESUMO
Progressive Multifocal Leukoencephalopathy (PML) is a demyelinating brain disease caused by human polyoma JC virus (JCV). This disease is an important cause of morbidity and mortality in AIDS patients. Definite diagnosis currently requires a brain biopsy. PCR for JCV of CSF, an emerging diagnostic tool, has a high specificity for the diagnosis of PML in patients with characteristics on clinical and neuroradiological findings. The authors report a 36-year-old woman who presented with prolonged fever, progressive weakness, and slow speech for 2 months. Clinical features and MRI findings were compatible with PML. Qualitative PCR for JCV of CSF showed a positive result. This report emphasizes the yield of PCR, the CSF for JCV in a diagnosis of PML, which may reduce the need for a brain biopsy in such cases.
Assuntos
Síndrome da Imunodeficiência Adquirida/complicações , Vírus JC/isolamento & purificação , Leucoencefalopatia Multifocal Progressiva/diagnóstico , Síndrome da Imunodeficiência Adquirida/tratamento farmacológico , Adulto , Terapia Antirretroviral de Alta Atividade , Antivirais/uso terapêutico , Biópsia , Líquido Cefalorraquidiano/virologia , Feminino , Humanos , Leucoencefalopatia Multifocal Progressiva/complicações , Leucoencefalopatia Multifocal Progressiva/epidemiologia , Leucoencefalopatia Multifocal Progressiva/terapia , Procedimentos Neurocirúrgicos , Reação em Cadeia da Polimerase , Tailândia/epidemiologia , Resultado do TratamentoRESUMO
The aim of this study was to determine the performance of real-time amplification based methods - NASBA, TaqMan, RT-FRET, and RT-PCR LUXtrade mark formats - for the detection of influenza A (H5N1) virus RNA. In an analysis of 54 samples obtained from a range of animal species in Thailand during the period 2003-2006, results showed that the NASBA (H5=98.2%, N1=96.3%), TaqMan (H5=98.2%, N1=96.3%) and FRET (H5=98.2%, N1=96.3%) had significantly higher rates of positive detection than LUX (H5=94.4%, N1=50.0%; P<0.001) for influenza A, H5 and N1 isolates. There were no false-positive results from any methods used in the negative-control group of samples. The limits of analytical detection were at least 10copies/reaction in real-time NASBA and LUX assays, while FRET and TaqMan assay appeared to be less sensitive at > or =100copies/reaction. The assays were relatively specific without cross-reactivity to a number of other influenza strains or viral pathogens. In conclusion, our study demonstrated that real-time NASBA, TaqMan and FRET assays can be used to detect influenza A (H5N1) from a wide range of hosts, and be specific for H5N1 samples obtained during different outbreaks (2003-2006). All assays provided the benefit of rapid influenza H5N1 identification for early diagnosis, in the range of hours, and they are well suited to high throughput analyses.