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1.
BMC Infect Dis ; 10: 262, 2010 Sep 07.
Artículo en Inglés | MEDLINE | ID: mdl-20822507

RESUMEN

BACKGROUND: The number of HIV-1 infected individuals in the Western world continues to rise. More in-depth understanding of regional HIV-1 epidemics is necessary for the optimal design and adequate use of future prevention strategies. The use of a combination of phylogenetic analysis of HIV sequences, with data on patients' demographics, infection route, clinical information and laboratory results, will allow a better characterization of individuals responsible for local transmission. METHODS: Baseline HIV-1 pol sequences, obtained through routine drug-resistance testing, from 506 patients, newly diagnosed between 2001 and 2009, were used to construct phylogenetic trees and identify transmission-clusters. Patients' demographics, laboratory and clinical data, were retrieved anonymously. Statistical analysis was performed to identify subtype-specific and transmission-cluster-specific characteristics. RESULTS: Multivariate analysis showed significant differences between the 59.7% of individuals with subtype B infection and the 40.3% non-B infected individuals, with regard to route of transmission, origin, infection with Chlamydia (p = 0.01) and infection with Hepatitis C virus (p = 0.017). More and larger transmission-clusters were identified among the subtype B infections (p < 0.001). Overall, in multivariate analysis, clustering was significantly associated with Caucasian origin, infection through homosexual contact and younger age (all p < 0.001). Bivariate analysis additionally showed a correlation between clustering and syphilis (p < 0.001), higher CD4 counts (p = 0.002), Chlamydia infection (p = 0.013) and primary HIV (p = 0.017). CONCLUSIONS: Combination of phylogenetics with demographic information, laboratory and clinical data, revealed that HIV-1 subtype B infected Caucasian men-who-have-sex-with-men with high prevalence of sexually transmitted diseases, account for the majority of local HIV-transmissions. This finding elucidates observed epidemiological trends through molecular analysis, and justifies sustained focus in prevention on this high risk group.


Asunto(s)
Infecciones por VIH/epidemiología , Infecciones por VIH/virología , VIH-1/clasificación , VIH-1/genética , Filogenia , ARN Viral/genética , Adulto , Infecciones por Chlamydia/epidemiología , Análisis por Conglomerados , Comorbilidad , Femenino , Genotipo , VIH-1/aislamiento & purificación , Hepatitis C/epidemiología , Homosexualidad Masculina , Humanos , Masculino , Epidemiología Molecular , Prevalencia , Análisis de Secuencia de ADN , Homología de Secuencia , Población Blanca , Productos del Gen pol del Virus de la Inmunodeficiencia Humana/genética
2.
J Clin Virol ; 106: 58-63, 2018 09.
Artículo en Inglés | MEDLINE | ID: mdl-30077957

RESUMEN

BACKGROUND: Successfully treated HIV-1 infected patients have a sustained undetectable viral RNA load. In these cases the total HIV-1 DNA load may constitute a valuable tool to further follow the overall viral burden. The value of this marker outside of cure research has been rarely studied. OBJECTIVES: To develop a quantitative (q)PCR for total HIV-1 DNA quantification in buffy coat cells and to evaluate the value of this parameter in clinical follow-up. STUDY DESIGN: A qPCR using primers and a probe in the conserved HIV-1 LTR region was adapted for use on DNA extracted from buffy coat cells. Sensitivity, accuracy and reproducibility were evaluated using 8E5 cells and samples from naive and treatment experienced patients. The clinical value of DNA load analysis was assessed by testing 119 longitudinal samples from 9 patients before and after ART initiation and 249 cross sectional samples from therapy-experienced patients. RESULTS: Inter- and intra-assay coefficients of variability were 5.56 and 5.94 (%CV). HIV-1 DNA was detected in 249 of the 263 (94.7%) patients on ART for at least 5 months (median: 53 months; IQR: 28-84 months). The HIV-1 DNA load varied between 0.60 and 3.37 copies/106 blood cells and showed significant correlation with the pre-ART CD4+ T-cell count nadir and peak viral RNA load. ART initiation resulted in a slow and limited decline of the total HIV-1 DNA concentration. CONCLUSIONS: Quantification of total HIV-1 DNA from buffy coat cells is feasible, sensitive and reliable. Although determination of the on-therapy HIV-1 DNA load may be informative, regular testing has limited clinical value because of the very slow evolution.


Asunto(s)
Capa Leucocitaria de la Sangre/virología , ADN Viral/análisis , Infecciones por VIH/tratamiento farmacológico , Carga Viral/métodos , Adulto , Fármacos Anti-VIH/uso terapéutico , Terapia Antirretroviral Altamente Activa , Recuento de Linfocito CD4 , Estudios Transversales , Cartilla de ADN/genética , Estudios de Seguimiento , Marcadores Genéticos , Infecciones por VIH/epidemiología , Seropositividad para VIH , VIH-1/genética , Humanos , Estudios Longitudinales , Masculino , Persona de Mediana Edad , ARN Viral , Reacción en Cadena en Tiempo Real de la Polimerasa , Reproducibilidad de los Resultados , Estudios Retrospectivos
3.
Antivir Ther ; 23(3): 277-281, 2018.
Artículo en Inglés | MEDLINE | ID: mdl-29063859

RESUMEN

BACKGROUND: Pre-analytical sample processing is often overlooked as a potential cause of inaccurate assay results. Here we demonstrate how plasma, extracted from standard EDTA-containing blood collection tubes, may contain traces of blood cells consequently resulting in a false low-level HIV-1 viral load when using Roche Cobas HIV-1 assays. METHODS: The presence of human DNA in Roche Cobas 4800 RNA extracts and in RNA extracts from the Abbott HIV-1 RealTime assay was assessed by quantifying the human albumin gene by means of quantitative PCR. RNA was extracted from plasma samples before and after an additional centrifugation and tested for viral load and DNA contamination. The relation between total DNA content and viral load was defined. RESULTS: Elevated concentrations of genomic DNA were detected in 28 out of 100 Cobas 4800 extracts and were significantly more frequent in samples processed outside of the AIDS Reference Laboratory. An association between genomic DNA presence and spurious low-level viraemia results was demonstrated. Supplementary centrifugation of plasma before RNA extraction eliminated the contamination and the false viraemia. CONCLUSIONS: Plasma isolated from standard EDTA-containing blood collection tubes may contain traces of HIV DNA leading to false viral load results above the clinical cutoff. Supplementary centrifugation of plasma before viral load analysis may eliminate the occurrence of this spurious low-level viraemia.


Asunto(s)
Infecciones por VIH/diagnóstico , Infecciones por VIH/virología , VIH-1 , Carga Viral , Viremia/virología , Bioensayo/métodos , Bioensayo/normas , Contaminación de ADN , VIH-1/genética , Humanos , ARN Viral , Juego de Reactivos para Diagnóstico , Reacción en Cadena en Tiempo Real de la Polimerasa/métodos , Reacción en Cadena en Tiempo Real de la Polimerasa/normas , Sensibilidad y Especificidad
4.
Virology ; 510: 185-193, 2017 10.
Artículo en Inglés | MEDLINE | ID: mdl-28750322

RESUMEN

HIV-infected patients on antiretroviral therapy (ART) may present low-level viremia (LLV) above the detection level of current viral load assays. In many cases LLV is persistent but does not result in overt treatment failure or selection of drug resistant viral variants. To elucidate whether LLV reflects active virus replication, we extensively sequenced pol and env genes of the viral populations present before and during LLV in 18 patients and searched for indications of genetic evolution. Maximum likelihood phylogenetic trees were inspected for temporal structure both visually and by linear regression analysis of root-to-tip and pairwise distances. Viral coreceptor tropism was assessed at different time points before and during LLV. In none of the patients consistent indications for genetic evolution were found over a median period of 4.8 years of LLV. As such these findings could not provide evidence that active virus replication is the main driver of LLV.


Asunto(s)
Antirretrovirales/uso terapéutico , Evolución Molecular , Infecciones por VIH/tratamiento farmacológico , Infecciones por VIH/virología , VIH-1/genética , VIH-1/aislamiento & purificación , VIH-1/fisiología , Humanos , Estudios Longitudinales , Filogenia , Análisis de Secuencia de ADN , Tropismo Viral , Productos del Gen env del Virus de la Inmunodeficiencia Humana/genética , Productos del Gen pol del Virus de la Inmunodeficiencia Humana/genética
5.
Virology ; 426(1): 7-11, 2012 Apr 25.
Artículo en Inglés | MEDLINE | ID: mdl-22305619

RESUMEN

There are conflicting data on the impact of low frequency HIV-1 drug-resistant mutants on the response of first-line highly active antiretroviral therapy (HAART), more specifically containing a NNRTI. As population sequencing does not detect resistant viruses representing less than 15-25% of the viral population, more sensitive techniques have been developed but still need clinical validation. We evaluated ultra-deep sequencing (UDPS), recently more available and affordable, as a tool for the detection of HIV-1 minority species carrying drug resistant mutation (DRM) in a clinical setting. A retrospective analysis of the reverse transcriptase (RT) gene of plasma HIV-1 from 70 patients starting a NNRTI based regimen was performed. Minority populations were defined as representing > 1% and < 20% of the total viral population. Using UDPS, we could not confirm an association between the presence of low minority variants harbouring RT mutations at the start of therapy and primary or secondary therapeutic failure.


Asunto(s)
Fármacos Anti-VIH/uso terapéutico , Infecciones por VIH/tratamiento farmacológico , Transcriptasa Inversa del VIH/genética , VIH-1/enzimología , VIH-1/aislamiento & purificación , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Inhibidores de la Transcriptasa Inversa/uso terapéutico , Adulto , Terapia Antirretroviral Altamente Activa , Farmacorresistencia Viral , Femenino , Infecciones por VIH/virología , Transcriptasa Inversa del VIH/antagonistas & inhibidores , VIH-1/clasificación , VIH-1/genética , Humanos , Masculino , Persona de Mediana Edad , Mutación , Estudios Retrospectivos
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