Stable hepatitis C virus RNA detection by RT-PCR during four days storage

BACKGROUND: Suboptimal specimen processing and storage conditions of samples which contain hepatitis C virus (HCV) RNA may result in a decline of HCV RNA concentration or false-negative results in the detection of HCV RNA in serum. We evaluated the stability of HCV RNA in serum and clotted blood samples stored at room temperature or at 4°C for 4 days with the aim of optimizing the standard procedures of processing and storage of samples. METHODS: Blood from five HCV RNA positive patients was collected in tubes with and without separator gel, centrifuged 1 or 6 hours after collection. Samples were then left 6, 24, 48, 72 or 96 h at room temperature (21.5 – 25.4°C) or at 4°C before determining their HCV RNA level using the COBAS AMPLICOR HCV MONITOR Test, vs 2.0 (Roche Diagnostic Systems). RESULTS: The logarithm of the HCV RNA level measurements remained within a 0.3 value of the means for 4 days at both temperatures (room temperature or 4°C). CONCLUSIONS: We conclude that blood samples may be collected and aliquoted within 6 h of collection and can be stored at 4°C for 72 hours as proposed by the manufacturer without significant differences in measured HCV RNA level. Our results indicate that lapses in this scheme may still yield reliable results

HIV-1 Load Comparison Using Four Commercial Real-Time Assays

The HIV-1 RNA viral load is commonly used for the monitoring of disease progression and antiretroviral treatment of HIV-1-infected patients. Since the misestimating of values could lead to inappropriate therapeutical management, the comparative performances, especially the ability to span the genetic diversity of HIV-1, of available automated real-time assays need to be evaluated. We conducted a prospective study with 74 consenting patients enrolled between March 2007 and November 2008. A blood sample was obtained at the time of diagnosis of HIV seropositivity and blindly tested for HIV-1 RNA by at least 4 commercial tests: the Abbott m2000 RealTime HIV-1, bioMérieux NucliSens EasyQ HIV-1, version 1.2 (v1.2), and Cobas AmpliPrep/Cobas TaqMan (CAP/CTM) v1.0 and v2.0 assays. The means of difference were null between CAP/CTM v2.0 and Abbott for CRF02_AG subtypes but positive in favor of CAP/CTM v2.0 for genotype B and negative in favor of NucliSens for all genotypes. The standard deviation (SD) of difference ranged from 0.3 to 0.59, depending on the considered couples of assays. Reliabilities of these four tests, appreciated by the standard deviation of difference between the measurement and the estimated “true” viral load and by the coefficient of reliability, were significantly different (P < 10(−4)) among each other. Significant differences were also observed within each group of HIV-1 genotype. The global disparity was higher for CRF02_AG than for B subtypes. This study indicates a risk of viral load misestimating or discrepancies between techniques, depending on the HIV-1 subtype, and speaks in favor of using the same assay for the monitoring of HIV-1-infected patients

Determinação do tropismo viral por ensaios genotípicos e\ud fenotípicos em pacientes brasileiros infectados por HIV-1

The clinical application of CCR5 antagonists involves first determining the coreceptor usage by the infecting viral strain.\ud Bioinformatics programs that predict coreceptor usage could provide an alternative method to screen candidates for treatment with\ud CCR5 antagonists, particularly in countries with limited financial resources. Thus, the present study aims to identify the best approach\ud using bioinformatics tools for determining HIV-1 coreceptor usage in clinical practice. Proviral DNA sequences and Trofile results\ud from 99 HIV-1-infected subjects under clinical monitoring were analyzed in this study. Based on the Trofile results, the viral variants\ud present were 81.1% R5, 21.4% R5X4 and 1.8% X4. Determination of tropism using a Geno2pheno\ud [coreceptor]\ud analysis with a false\ud positive rate of 10% gave the most suitable performance in this sampling: the R5 and X4 strains were found at frequencies of 78.5%\ud and 28.4%, respectively, and there was 78.6% concordance between the phenotypic and genotypic results. Further studies are needed\ud to clarify how genetic diversity amongst virus strains affects bioinformatics-driven approaches for determining tropism. Although this\ud strategy could be useful for screening patients in developing countries, some limitations remain that restrict the wider application of\ud coreceptor usage tests in clinical practice.A aplicação clínica dos antagonistas de CCR5 envolve em primeiro lugar determinar o uso de co-receptor pela cepa viral infectante. Programas de bioinformática que prevêem o uso co-receptor poderiam fornecer um método alternativo para selecionar candidatos para o tratamento com os antagonistas do CCR5, particularmente em países com poucos recursos financeiros. Assim, o presente estudo teve por objetivo identificar a melhor abordagem utilizando ferramentas de bioinformática para determinar qual o tipo de co-receptor do HIV-1 que poderia ser usado na prática clínica. Sequências de DNA proviral e Trofile resultados a partir de 99 pacientes infectados pelo HIV-1 sob monitorização clínica foram avaliadas. Com base nos resultados do Teste Trofile, as variantes virais presentes eram R5 (81,1%), R5X4 (21,4%) e X4 (1,8%). Determinação do tropismo pela análise do Geno2pheno, com taxa de falso positivos de 10% apresentou desempenho mais adequado para esta amostragem: as cepas R5 e X4 foram encontradas em frequências de 78,5% e 28,4%, respectivamente, e foi de 78,6% a concordância entre os resultados fenotípicos e genotípicos. Mais estudos são necessários para esclarecer como a diversidade genética entre as cepas do vírus afeta abordagens baseadas na determinação do tropismo pelas ferramentas de bioinformática. Embora esta estratégia possa ser útil para o rastreio de pacientes em países em desenvolvimento, permanecem algumas limitações que restringem a aplicação mais ampla para utilização de testes de co-receptor na prática clínica.The financial support from FAPESP (08/58138-0; 08/51265-6; 10/00222-5); FFM; CNPq