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The persistence of a reservoir of latently infected CD4 T cells remains one of the major obstacles to cure HIV. Numerous strategies are being explored to eliminate this reservoir. To translate these efforts into clinical trials, there is a strong need for validated biomarkers that can monitor the reservoir over time in vivo. A comprehensive study was designed to evaluate and compare potential HIV-1 reservoir biomarkers. A cohort of 25 patients, treated with suppressive antiretroviral therapy was sampled at three time points, with median of 2.5 years (IQR: 2.4-2.6) between time point 1 and 2; and median of 31 days (IQR: 28-36) between time point 2 and 3. Patients were median of 6 years (IQR: 3-12) on ART, and plasma viral load (<50 copies/ml) was suppressed for median of 4 years (IQR: 2-8). Total HIV-1 DNA, unspliced (us) and multiply spliced HIV-1 RNA, and 2LTR circles were quantified by digital PCR in peripheral blood, at 3 time points. At the second time point, a viral outgrowth assay (VOA) was performed, and integrated HIV-1 DNA and relative mRNA expression levels of HIV-1 restriction factors were quantified. No significant change was found for long- and short-term dynamics of all HIV-1 markers tested in peripheral blood. Integrated HIV-1 DNA was associated with total HIV-1 DNA (p<0.001, R² = 0.85), us HIV-1 RNA (p = 0.029, R² = 0.40), and VOA (p = 0.041, R2 = 0.44). Replication-competent virus was detected in 80% of patients by the VOA and it correlated with total HIV-1 DNA (p = 0.039, R² = 0.54). The mean quantification difference between Alu-PCR and VOA was 2.88 log10, and 2.23 log10 between total HIV-1 DNA and VOA. The levels of usHIV-1 RNA were inversely correlated with mRNA levels of several HIV-1 restriction factors (TRIM5α, SAMHD1, MX2, SLFN11, pSIP1). Our study reveals important correlations between the viral outgrowth and total and integrated HIV-1 DNA measures, suggesting that the total pool of HIV-1 DNA may predict the size of the replication-competent virus in ART suppressed patients.
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Infecciones por VIH/virología , VIH-1/crecimiento & desarrollo , Integración Viral , Adulto , Estudios de Cohortes , ADN Viral/análisis , ADN Viral/aislamiento & purificación , Reservorios de Enfermedades/virología , Femenino , VIH-1/genética , Humanos , Modelos Lineales , Masculino , Persona de Mediana Edad , ARN Viral/análisis , ARN Viral/aislamiento & purificación , Carga ViralRESUMEN
UNLABELLED: The ability to persist long term in latently infected CD4 T cells represents a characteristic feature of HIV-1 infection and the predominant barrier to efforts aiming at viral eradication and cure. Yet, increasing evidence suggests that only small subsets of CD4 T cells with specific developmental and maturational profiles are able to effectively support HIV-1 long-term persistence. Here, we analyzed how the functional polarization of CD4 T cells shapes and structures the reservoirs of HIV-1-infected cells. We found that CD4 T cells enriched for a Th1/17 polarization had elevated susceptibilities to HIV-1 infection in ex vivo assays, harbored high levels of HIV-1 DNA in persons treated with antiretroviral therapy, and made a disproportionately increased contribution to the viral reservoir relative to their contribution to the CD4 T memory cell pool. Moreover, HIV-1 DNA levels in Th1/17 cells remained stable over many years of antiretroviral therapy, resulting in a progressively increasing contribution of these cells to the viral reservoir, and phylogenetic studies suggested preferential long-term persistence of identical viral sequences during prolonged antiretroviral treatment in this cell compartment. Together, these data suggest that Th1/17 CD4 T cells represent a preferred site for HIV-1 DNA long-term persistence in patients receiving antiretroviral therapy. IMPORTANCE: Current antiretroviral therapy is very effective in suppressing active HIV-1 replication but does not fully eliminate virally infected cells. The ability of HIV-1 to persist long term despite suppressive antiretroviral combination therapy represents a perplexing aspect of HIV-1 disease pathogenesis, since most HIV-1 target cells are activated, short-lived CD4 T cells. This study suggests that CD4 T helper cells with Th1/17 polarization have a preferential role as a long-term reservoir for HIV-1 infection during antiretroviral therapy, possibly because these cells may imitate some of the functional properties traditionally attributed to stem cells, such as the ability to persist for extremely long periods of time and to repopulate their own pool size through homeostatic self-renewal. These observations support the hypothesis that HIV-1 persistence is driven by small subsets of long-lasting stem cell-like CD4 T cells that may represent particularly promising targets for clinical strategies aiming at HIV-1 eradication and cure.
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Fármacos Anti-VIH/uso terapéutico , Polaridad Celular/inmunología , Infecciones por VIH/tratamiento farmacológico , Células TH1/inmunología , Células Th17/inmunología , Latencia del Virus/genética , Adulto , Secuencia de Bases , Recuento de Linfocito CD4 , Células Cultivadas , ADN Viral/genética , Femenino , Infecciones por VIH/virología , VIH-1/genética , VIH-1/inmunología , Humanos , Masculino , Persona de Mediana Edad , Análisis de Secuencia de ADN , Células TH1/virología , Células Th17/virología , Células Th2/inmunología , Células Th2/virología , Replicación Viral/genéticaRESUMEN
Episomal HIV-1 two-long terminal repeat (2-LTR) circles are considered markers for ongoing viral replication. Two sample processing procedures were compared to accurately quantify 2-LTR in patients by using droplet digital PCR (ddPCR). Here, we show that plasmid isolation with a spiked non-HIV plasmid for normalization enables more accurate 2-LTR quantification than genomic DNA isolation.
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ADN Viral/aislamiento & purificación , VIH-1/genética , Plásmidos/aislamiento & purificación , Reacción en Cadena de la Polimerasa/métodos , Secuencias Repetidas Terminales , ADN Viral/análisis , Humanos , Plásmidos/análisisRESUMEN
OBJECTIVES: Whether ART regimens differ in their propensity to allow persistent HIV-1 detection remains unclear. To investigate this, we performed a cross-sectional study to characterize HIV-1 persistence in peripheral blood during suppressive therapy with NRTIs plus a PI or nevirapine. METHODS: Residual plasma HIV-1 RNA was quantified by real-time PCR. Cell-associated proviral total HIV-1 DNA, unspliced and multiply spliced HIV-1 RNA and 2-long terminal repeat (2-LTR) circles were quantified by digital PCR. RESULTS: Comparing PI with nevirapine recipients, residual plasma HIV-1 RNA detection rates were 47/80 (58.8%) versus 37/81 (45.7%), with median (IQR) levels of 4 (3-8) versus 4 (3-7) copies/mL (Pâ=â0.207); detection was less likely with longer duration of suppressive ART (Pâ=â0.020), independently of treatment. HIV-1 DNA was detected in all patients, with median levels of 2.3 (IQR 2.0-2.7) versus 2.5 (IQR 2.1-2.7) log10 copies/10(6) PBMCs, respectively; HIV-1 DNA levels were associated with pre-ART viral load (Pâ=â0.004) and with residual HIV-1 RNA (Pâ=â0.034), unspliced HIV-1 RNA (Pâ=â0.001) and 2-LTR circles (Pâ=â0.005), independently of treatment. CONCLUSIONS: No significant differences were revealed in levels of residual plasma HIV-1 RNA, total HIV-1 DNA or intracellular markers of ongoing virus replication (unspliced and multiply spliced HIV-1 RNA and 2-LTR circles) between treatment groups.
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Fármacos Anti-VIH/uso terapéutico , Terapia Antirretroviral Altamente Activa/métodos , ADN Viral/sangre , Infecciones por VIH/tratamiento farmacológico , VIH-1/aislamiento & purificación , ARN Viral/sangre , Carga Viral , Adulto , Estudios Transversales , Femenino , Inhibidores de la Proteasa del VIH/uso terapéutico , Humanos , Masculino , Persona de Mediana Edad , Nevirapina/uso terapéutico , Reacción en Cadena de la Polimerasa , Resultado del TratamientoRESUMEN
Digital PCR is rapidly gaining interest in the field of molecular biology for absolute quantification of nucleic acids. However, the first generation of platforms still needs careful validation and requires a specific methodology for data analysis to distinguish negative from positive signals by defining a threshold value. The currently described methods to assess droplet digital PCR (ddPCR) are based on an underlying assumption that the fluorescent signal of droplets is normally distributed. We show that this normality assumption does not likely hold true for most ddPCR runs, resulting in an erroneous threshold. We suggest a methodology that does not make any assumptions about the distribution of the fluorescence readouts. A threshold is estimated by modelling the extreme values in the negative droplet population using extreme value theory. Furthermore, the method takes shifts in baseline fluorescence between samples into account. An R implementation of our method is available, allowing automated threshold determination for absolute ddPCR quantification using a single fluorescent reporter.
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Reacción en Cadena de la Polimerasa/métodos , FluorescenciaRESUMEN
The central memory T cell (TCM) model forms a unique HIV-1 latency model based on primary cells that closely resemble in vivo TCM. The virus employed in this model is based on an engineered vector incapable of replication after initial infection. We show that despite this strategy, replication competent viral particles are released into the culture medium due to recombination between overlapping sequences of the env deleted HIV genome that is co-transfected with intact env. This finding emphasizes the need for careful data analysis and interpretation if similar constructs are employed and urges for additional caution during laboratory work.
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Infecciones por VIH/virología , VIH-1/fisiología , Latencia del Virus , Replicación Viral/genética , Linfocitos T CD4-Positivos/virología , ADN Viral/genética , Genes env , Vectores Genéticos/genética , VIH-1/genética , HumanosRESUMEN
Digital polymerase chain reaction (PCR) is an emerging absolute quantification method based on the limiting dilution principle and end-point PCR. This methodology provides high flexibility in assay design without influencing quantitative accuracy. This article describes an assay to quantify HIV DNA that targets a highly conserved region of the HIV-1 genome that hampers optimal probe design. To maintain high specificity and allow probe binding and hydrolysis of a probe with low melting temperature, a two-stage touchdown PCR was designed with a first round of amplification at high temperature and a subsequent round at low temperature to allow accumulation of fluorescence.
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Secuencia Conservada , Genoma Viral , VIH-1/genética , Reacción en Cadena de la Polimerasa/métodos , ARN Viral/genética , Secuencia de BasesRESUMEN
Toxoplasma gondii, an obligate intracellular parasitic protozoon, usually causes a mild, acute infection followed by a latent asymptomatic phase with tissue cysts or a chronic form with recurrent retinochoroiditis. However, immunocompromised patients can cause disseminated disease due to the reactivation of the latent tissue cysts or due to a primary infection. Here, we present a rare case of bilateral ocular toxoplasmosis and concurrent subacute toxoplasma encephalitis in a 70-year-old patient on anti-CD40 treatment following his liver transplant. The diagnosis was confirmed by PCR of anterior chamber fluid and brain biopsy, and no other sites of disseminated disease were detected on PET-CT. The patient has been treated with sulfamethoxazole-trimethoprim 800/160 mg with virtually complete resolution of the neurological and ocular symptoms. Iatrogenic blockade of the CD40 pathway may elicit a particular susceptibility for CNS reactivation of T. gondii.
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Extrapulmonary nontuberculous mycobacterial (NTM) disease remains largely enigmatic, yet these mycobacteria are increasingly acknowledged as important opportunistic pathogens in humans. Traditionally, NTM infections have been identified across various anatomical locations, with the respiratory system being the most affected and best understood. Historically, extrapulmonary NTM infection was predominantly associated with HIV/AIDS, with Mycobacterium avium lymphadenopathy being the most commonly reported. Today, however, because of the expanding utilization of immunosuppressive therapies and the demographic shift towards an aging population, an increasing number of NTM infections are expected and seen. Hence, a heightened index of suspicion is essential, necessitating a multifaceted approach to identification and drug sensitivity testing to improve treatment outcomes. In extrapulmonary NTM management, expert consultation is strongly recommended to determine the most efficacious treatment regimen, as individualized, patient-tailored therapies are often required. Furthermore, the economic burden of NTM disease is considerable, accompanied by high rates of hospitalization. To optimize the management of these intricate infections, there is an urgent need for comprehensive data on incidence, prevalence, and outcomes. This case-based series delves into the intricate nature of extrapulmonary NTM infections, focusing on both rapid and slow-growing NTM species, and explores therapeutic options, resistance mechanisms, and host-related immunological factors.
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BACKGROUND: Because of the magnitude of the global tobacco epidemic, the World Health Organisation developed the Framework Convention on Tobacco Control (FCTC), an international legally binding treaty to control tobacco use. Adoption and implementation of specific tobacco control measures within FCTC is an outcome of a political process, where social norms and public opinion play important roles. The objective of our study was to examine how a country's level of tobacco control is associated with smoking prevalence, two markers of denormalisation of smoking (social disapproval of smoking and concern about passive smoking), and societal support for tobacco control. METHODS: An ecological study was conducted, using data from two sources. The first source was the Tobacco Control Scale (TCS) from 2011, which quantifies the implementation of tobacco control policies in European Union (EU) countries. Data on smoking prevalence, societal disapproval of smoking, concern about passive smoking, and societal support for policy measures were taken from the Eurobarometer survey of 2009. Data from Eurobarometer surveys were aggregated to country level. Data from the 27 European Union member states were used. RESULTS: Smoking prevalence rates in 2009 were negatively associated with a country's TCS 2011 score, although not statistically significant (r = -.25; p = .21). Experience of societal disapproval was positively associated with higher TCS scores, though not significantly (r = .14; p = .48). The same was true for societal support for tobacco control (r = .27; p = .18). The TCS score in 2011 was significantly correlated with concern about passive smoking (r = .42; p =.03). Support for tobacco control measures was also strongly correlated with concern about passive smoking (r = .52, p = .006). CONCLUSIONS: Smokers in countries with a higher TCS score were more concerned about whether their smoke harms others. Further, support for tobacco control measures is higher in countries that have more of these concerned smokers. Concerns about passive smoking seem central in the implementation of tobacco control measures, stressing the importance of continuing to educate the public about the harm from passive smoking.
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Actitud Frente a la Salud , Política de Salud , Prevención del Hábito de Fumar , Contaminación por Humo de Tabaco , Europa (Continente)/epidemiología , Humanos , Prevalencia , Fumar/epidemiología , Valores Sociales , Contaminación por Humo de Tabaco/prevención & controlRESUMEN
INTRODUCTION: Anaplastic lymphoma kinase (ALK) gene translocation occurs in 3%-5% of patients with non-small cell lung cancer (NSCLC), typically in younger patients. Crizotinib (tyrosine kinase inhibitor) has been considered as the standard of care for advanced ALK-positive lung cancer but it only gives a median progression-free survival of 7.7-11 months. CASE: A 41-year-old old man, former smoker, was diagnosed with NSCLC in the right lung with manifest pleural effusion. This case was complicated by a pleural empyema and because of a trapped lung, there was an indication for the construction of a thoracostomy. After confirmation of the ALK translocation, therapy with crizotinib was started. After 8 weeks, there was excellent response, and 6 months later, all lesions were undetectable on CT scan. There was also complete healing of the thoracostomy wound. CONCLUSION: This case describes a relatively young patient with a poor prognosis but with a remarkable and long-term response to crizotinib monotherapy.
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Quinasa de Linfoma Anaplásico/genética , Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Carcinoma de Pulmón de Células no Pequeñas/genética , Crizotinib/uso terapéutico , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/genética , Inhibidores de Proteínas Quinasas/uso terapéutico , Adulto , Carcinoma de Pulmón de Células no Pequeñas/complicaciones , Carcinoma de Pulmón de Células no Pequeñas/diagnóstico , Crizotinib/administración & dosificación , Crizotinib/efectos adversos , Empiema/complicaciones , Empiema/diagnóstico , Empiema/cirugía , Humanos , Neoplasias Pulmonares/complicaciones , Neoplasias Pulmonares/diagnóstico , Masculino , Inhibidores de Proteínas Quinasas/administración & dosificación , Inhibidores de Proteínas Quinasas/efectos adversos , Radiografía Torácica , Toracostomía , Tomografía Computarizada por Rayos X , Resultado del TratamientoRESUMEN
HIV-1 DNA quantification serves as an important reservoir biomarker in HIV cure trials. However, the high genetic diversity of HIV-1 represented by different subtypes may bring inaccuracy in quantifying HIV-1 DNA and a sensitive and validated assay covering diverse HIV-1 subtypes is lacking. Therefore, we cross-validated total HIV-1 DNA assays described in literature using a three-step comparative analysis. First, a bioinformatics tool was developed in-house to perform an in silico evaluation of 67 HIV-1 DNA assays. Secondly, these selected assays were in vitro validated using a panel of different HIV-1 subtypes and, finally, ex vivo assessed on selected patient samples with different HIV-1 subtypes. Our results show that quantification of HIV-1 DNA substantially differs between assays and we advise five best performing HIV-1 DNA assays for ddPCR and qPCR (Schvachsa_2007, Viard_2004, Heeregrave_2009, Van_der_Sluis_2013, Yu_2008 and Yun_2002). This in-depth analysis of published HIV-1 DNA assays indicates that not all assays guarantee an optimal measurement of HIV-1 DNA, especially when looking across subtypes. Using an in-depth cross-validation, we were able to validate HIV-1 DNA assays that are suitable for quantification of HIV-1 DNA in a wide variety of HIV-1 infected patients.
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Infecciones por VIH/virología , VIH-1/genética , Reacción en Cadena de la Polimerasa/métodos , Simulación por Computador , ADN Viral/análisis , Variación Genética , Infecciones por VIH/genética , Duplicado del Terminal Largo de VIH , Humanos , Juego de Reactivos para Diagnóstico , Carga ViralRESUMEN
Reactivation of latent viral reservoirs is on the forefront of HIV-1 eradication research. However, it is unknown if latency reversing agents (LRAs) increase the level of viral transcription from cells producing HIV RNA or harboring transcriptionally-inactive (latent) infection. We therefore developed a microfluidic single-cell-in-droplet (scd)PCR assay to directly measure the number of CD4+ T cells that produce unspliced (us)RNA and multiply spliced (ms)RNA following ex vivo latency reversal with either an histone deacetylase inhibitor (romidepsin) or T cell receptor (TCR) stimulation. Detection of HIV-1 transcriptional activity can also be performed on hundreds of thousands of CD4+ T-cells in a single experiment. The scdPCR method was then applied to CD4+ T cells obtained from HIV-1-infected individuals on antiretroviral therapy. Overall, our results suggest that effects of LRAs on HIV-1 reactivation may be heterogeneous-increasing transcription from active cells in some cases and increasing the number of transcriptionally active cells in others. Genomic DNA and human mRNA isolated from HIV-1 reactivated cells could also be detected and quantified from individual cells. As a result, our assay has the potential to provide needed insight into various reservoir eradication strategies.
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Infecciones por VIH/virología , VIH-1/genética , Ensayos Analíticos de Alto Rendimiento , Reacción en Cadena de la Polimerasa , ARN Viral , Análisis de la Célula Individual , Latencia del Virus , Adulto , Linfocitos T CD4-Positivos/virología , Células Cultivadas , Infecciones por VIH/tratamiento farmacológico , Humanos , Persona de Mediana Edad , Análisis de Secuencia de ADN , Carga Viral , Activación Viral/genéticaRESUMEN
Quantitative real-time PCR (qPCR) is implemented in many molecular laboratories worldwide for the quantification of viral nucleic acids. However, over the last two decades, there has been renewed interest in the concept of digital PCR (dPCR) as this platform offers direct quantification without the need for standard curves, a simplified workflow and the possibility to extend the current detection limit. These benefits are of great interest in terms of the quantification of low viral levels in HIV reservoir research because changes in the dynamics of residual HIV reservoirs will be important to monitor HIV cure efforts. Here, we have implemented a systematic literature screening and text mining approach to map the use of droplet dPCR (ddPCR) in the context of HIV quantification. In addition, several technical aspects of ddPCR were compared with qPCR: accuracy, sensitivity, precision and reproducibility, to determine its diagnostic utility. We have observed that ddPCR was used in different body compartments in multiple HIV-1 and HIV-2 assays, with the majority of reported assays focusing on HIV-1 DNA-based applications (i.e. total HIV DNA). Furthermore, ddPCR showed a higher accuracy, precision and reproducibility, but similar sensitivity when compared to qPCR due to reported false positive droplets in the negative template controls with a need for standardised data analysis (i.e. threshold determination). In the context of a low level of detection and HIV reservoir diagnostics, ddPCR can offer a valid alternative to qPCR-based assays but before this platform can be clinically accredited, some remaining issues need to be resolved.
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The mechanisms for the establishment and the persistence of the latent HIV-1 reservoir remain to be completely defined. HIV-1 infection is characterised by the integration of the reverse transcribed proviral DNA into the host's genome. This integrated proviral DNA can remain replication silent, but a small part of it is fully competent to restart viral replication when treatment is interrupted. Hence, this replication-competent provirus is the cause of viral rebound and is called the viral reservoir. The exact site of proviral integration within the host's cellular chromosome may affect the transcriptional activity of HIV. Thanks to recent technological advances, HIV-1 integration site analysis has been used to assess HIV-1 reservoirs in HIV-infected individuals. Analysis of HIV-1 integration sites in infected individuals undergoing suppressive ART led to identification of expanded clonal cell populations, indicating that clonal proliferation of the proviral reservoir may contribute to the long-term persistence of viral reservoirs. Here we describe the findings of several clinical studies, where a comprehensive HIV-1 integration site analysis was performed.
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BACKGROUND: Characterising the correlates of HIV persistence improves understanding of disease pathogenesis and guides the design of curative strategies. This study investigated factors associated with integrated HIV-1 DNA load during consistently suppressive first-line antiretroviral therapy (ART). METHOD: Total, integrated, and 2-long terminal repeats (LTR) circular HIV-1 DNA, residual plasma HIV-1 RNA, T-cell activation markers, and soluble CD14 (sCD14) were measured in peripheral blood of 50 patients that had received 1-14 years of efavirenz-based or nevirapine-based therapy. RESULTS: Integrated HIV-1 DNA load (per 10(6) peripheral blood mononuclear cells) was median 1.9 log10 copies (interquartile range 1.7-2.2) and showed a mean difference of 0.2 log10 copies per 10 years of suppressive ART (95% confidence interval - 0.2, 0.6; p = 0.28). It was positively correlated with total HIV-1 DNA load and frequency of CD8(+)HLA-DR/DP/DQ(+) cells, and was also higher in subjects with higher sCD14 levels, but showed no correlation with levels of 2-LTR circular HIV-1 DNA and residual plasma HIV-1 RNA, or the frequency of CD4(+)CD38(+) and CD8(+)CD38(+) cells. Adjusting for pre-ART viral load, duration of suppressive ART, CD4 cell counts, residual plasma HIV-1 RNA levels, and sCD14 levels, integrated HIV-1 DNA load was mean 0.5 log10 copies higher for each 50% higher frequency of CD8(+)HLA-DR/DP/DQ(+) cells (95% confidence interval 0.2, 0.9; p = 0.01). CONCLUSIONS: The observed positive association between integrated HIV-1 DNA load and frequency of CD8(+)DR/DP/DQ(+) cells indicates that a close correlation between HIV persistence and immune activation continues during consistently suppressive therapy. The inducers of the distinct activation profile warrant further investigation.
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Linfocitos T CD8-positivos/inmunología , ADN Viral/sangre , VIH-1/inmunología , Antígenos HLA-DP/inmunología , Antígenos HLA-DQ/inmunología , Antígenos HLA-DR/inmunología , Carga Viral/inmunología , Adulto , Terapia Antirretroviral Altamente Activa , Femenino , Humanos , Modelos Lineales , Masculino , Persona de Mediana Edad , Análisis MultivarianteRESUMEN
Persistent reservoirs remain the major obstacles to achieve an HIV-1 cure. Prolonged early antiretroviral therapy (ART) may reduce the extent of reservoirs and allow for virological control after ART discontinuation. We compared HIV-1 reservoirs in a cross-sectional study using polymerase chain reaction-based techniques in blood and tissue of early-treated seroconverters, late-treated patients, ART-naïve seroconverters, and long-term non-progressors (LTNPs) who have spontaneous virological control without treatment. A decade of early ART reduced the total and integrated HIV-1 DNA levels compared with later treatment initiation, but not reaching the low levels found in LTNPs. Total HIV-1 DNA in rectal biopsies did not differ between cohorts. Importantly, lower viral transcription (HIV-1 unspliced RNA) and enhanced immune preservation (CD4/CD8), reminiscent of LTNPs, were found in early compared to late-treated patients. This suggests that early treatment is associated with some immunovirological features of LTNPs that may improve the outcome of future interventions aimed at a functional cure.
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Antirretrovirales/uso terapéutico , Terapia Antirretroviral Altamente Activa/métodos , Infecciones por VIH/tratamiento farmacológico , Infecciones por VIH/epidemiología , VIH-1/aislamiento & purificación , Carga Viral , Adulto , Sangre/virología , Estudios Transversales , Femenino , Humanos , Masculino , Persona de Mediana Edad , Provirus/aislamiento & purificación , Recto/virología , Prevención Secundaria , Resultado del TratamientoRESUMEN
Abacavir is a nucleoside reverse transcriptase inhibitor used as part of combination antiretroviral therapy in HIV-1-infected patients. Because this drug can cause a hypersensitivity reaction that is correlated with the presence of the HLA-B*57:01 allotype, screening for the presence of HLA-B*57:01 is recommended before abacavir initiation. Different genetic assays have been developed for HLA-B*57:01 screening, each with specific sensitivity, turnaround time and assay costs. Here, a new real-time PCR (qPCR) based analysis is described and compared to sequence specific primer PCR with capillary electrophoresis (SSP PCR CE) on 149 patient-derived samples, using sequence specific oligonucleotide hybridization combined with high resolution SSP PCR as gold standard. In addition to these PCR based methods, a complementary approach was developed using flow cytometry with an HLA-B17 specific monoclonal antibody as a pre-screening assay to diminish the number of samples for genetic testing. All three assays had a maximum sensitivity of >99. However, differences in specificity were recorded, i.e. 84.3%, 97.2% and >99% for flow cytometry, qPCR and SSP PCR CE respectively. Our data indicate that the most specific and sensitive of the compared methods is the SSP PCR CE. Flow cytometry pre-screening can substantially decrease the number of genetic tests for HLA-B*57:01 typing in a clinical setting.
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Didesoxinucleósidos/administración & dosificación , Hipersensibilidad a las Drogas/prevención & control , Infecciones por VIH/tratamiento farmacológico , Antígenos HLA-B/genética , Reacción en Cadena en Tiempo Real de la Polimerasa/métodos , Inhibidores de la Transcriptasa Inversa/administración & dosificación , Alelos , Terapia Antirretroviral Altamente Activa , Cartilla de ADN/síntesis química , Cartilla de ADN/genética , Didesoxinucleósidos/efectos adversos , Hipersensibilidad a las Drogas/etiología , Hipersensibilidad a las Drogas/genética , Hipersensibilidad a las Drogas/inmunología , Electroforesis Capilar , Citometría de Flujo , Expresión Génica , Pruebas Genéticas/métodos , Infecciones por VIH/genética , Infecciones por VIH/inmunología , Infecciones por VIH/virología , VIH-1/efectos de los fármacos , VIH-1/enzimología , VIH-1/genética , Antígenos HLA-B/inmunología , Humanos , Hibridación de Ácido Nucleico/métodos , Inhibidores de la Transcriptasa Inversa/efectos adversos , Sensibilidad y EspecificidadRESUMEN
Cell-associated (CA) HIV-1 RNA is considered a potential marker for assessment of viral reservoir dynamics and antiretroviral therapy (ART) response in HIV-infected patients. Recent studies employed sensitive seminested real-time quantitative (q)PCR to quantify CA HIV-1 RNA. Digital PCR has been recently described as an alternative PCR-based technique for absolute quantification with higher accuracy compared to qPCR. Here, a comparison was made between the droplet digital PCR (ddPCR) and the seminested qPCR for quantification of unspliced (us) and multiply spliced (ms) CA HIV-1 RNA. Synthetic RNA standards and CA HIV-1 RNA from infected patients on and off ART (Nâ=â34) were quantified with both methods. Correlations were observed between the methods both for serially diluted synthetic standards (usRNA: R2â=â0.97, msRNA: R2â=â0.92) and patient-derived samples (usRNA: R2â=â0.51, msRNA: R2â=â0.87). Seminested qPCR showed better quantitative linearity, accuracy and sensitivity in the quantification of synthetic standards than ddPCR, especially in the lower quantification ranges. Both methods demonstrated equally high detection rate of usRNA in patient samples on and off ART (91%), whereas ddPCR detected msRNA in larger proportion of samples from ART-treated patients (pâ=â0.13). We observed an average agreement between the methods for usRNA quantification in patient samples, albeit with a large standard deviation (biasâ=â0.05±0.75 log10). However, a bias of 0.94±0.36 log10 was observed for msRNA. No-template controls were consistently negative in the seminested qPCR, but yielded a positive ddPCR signal for some wells. Therefore, the false positive signals may have affected the detection power of ddPCR in this study. Digital PCR is promising for HIV nucleic acid quantification, but the false positive signals need further attention. Quantitative assays for CA HIV RNA have the potential to improve monitoring of patients on ART and to be used in clinical studies aimed at HIV eradication, but should be cross-validated by multiple laboratories prior to wider use.