Cellular HIV-1 DNA Levels in Drug Sensitive Strains Are Equivalent to Those in Drug Resistant Strains in Newly-Diagnosed Patients in Europe

Background HIV-1 genotypic drug resistance is an important threat to the success of antiretroviral therapy and transmitted resistance has reached 9% prevalence in Europe. Studies have demonstrated that HIV-1 DNA load in peripheral blood mononuclear cells (PBMC) have a predictive value for disease progression, independently of CD4 counts and plasma viral load. Methodology/Principal Findings Molecular-beacon-based real-time PCR was used to measure HIV-1 second template switch (STS) DNA in PBMC in newly-diagnosed HIV-1 patients across Europe. These patients were representative for the HIV-1 epidemic in the participating countries and were carrying either drug-resistant or sensitive viral strains. The assay design was improved from a previous version to specifically detect M-group HIV-1 and human CCR5 alleles. The findings resulted in a median of 3.32 log10HIV-1copies/106PBMC and demonstrated for the first time no correlation between cellular HIV-1 DNA load and transmitted drug-resistance. A weak association between cellular HIV-1 DNA levels with plasma viral RNA load and CD4+T-cell counts was also reconfirmed. Co-receptor tropism for 91% of samples, whether or not they conferred resistance, was CCR5. A comparison of pol sequences derived from RNA and DNA, resulted in a high similarity between the two. Conclusions/Significance An improved molecular-beacon-based real-time PCR assay is reported for the measurement of HIV-1 DNA in PBMC and has investigated the association between cellular HIV-1 DNA levels and transmitted resistance to antiretroviral therapy in newly-diagnosed patients from across Europe. The findings show no correlation between these two parameters, suggesting that transmitted resistance does not impact disease progression in HIV-1 infected individuals. The CCR5 co-receptor tropism predominance implies that both resistant and non-resistant strains behave similarly in early infection. Furth

HIV-1 Infection in Cyprus, the Eastern Mediterranean European Frontier: A Densely Sampled Transmission Dynamics Analysis from 1986 to 2012

Since HIV-1 treatment is increasingly considered an effective preventionstrategy, it is important to study local HIV-1 epidemics to formulate tailored preventionpolicies. The prevalence of HIV-1 in Cyprus was historically low until 2005. To investigatethe shift in epidemiological trends, we studied the transmission dynamics of HIV-1 in Cyprususing a densely sampled Cypriot HIV-1 transmission cohort that included 85 percent ofHIV-1-infected individuals linked to clinical care between 1986 and 2012 based on detailedclinical, epidemiological, behavioral and HIV-1 genetic information. Subtyping andtransmission cluster reconstruction were performed using maximum likelihood and Bayesianmethods, and the transmission chain network was linked to the clinical, epidemiological andbehavioral data. The results reveal that for the main HIV-1 subtype A1 and B sub-epidemics,young and drug-naïve HIV-1-infected individuals in Cyprus are driving the dynamics of thelocal HIV-1 epidemic. The results of this study provide a better understanding of thedynamics of the HIV-1 infection in Cyprus, which may impact the development of preventionstrategies. Furthermore, this methodology for analyzing densely sampled transmissiondynamics is applicable to other geographic regions to implement effective HIV-1 preventionstrategies in local settings

Immune-escape mutations and stop-codons in HBsAg develop in a large proportion of patients with chronic HBV infection exposed to anti-HBV drugs in Europe

Background: HBsAg immune-escape mutations can favor HBV-transmission also in vaccinated individuals, promote immunosuppression-driven HBV-reactivation, and increase fitness of drug-resistant strains. Stop-codons can enhance HBV oncogenic-properties. Furthermore, as a consequence of the overlapping structure of HBV genome, some immune-escape mutations or stop-codons in HBsAg can derive from drug-resistance mutations in RT. This study is aimed at gaining insight in prevalence and characteristics of immune-associated escape mutations, and stop-codons in HBsAg in chronically HBV-infected patients experiencing nucleos(t)ide analogues (NA) in Europe. Methods: This study analyzed 828 chronically HBV-infected European patients exposed to ≥ 1 NA, with detectable HBV-DNA and with an available HBsAg-sequence. The immune-associated escape mutations and the NA-induced immune-escape mutations sI195M, sI196S, and sE164D (resulting from drug-resistance mutation rtM204 V, rtM204I, and rtV173L) were retrieved from literature and examined. Mutations were defined as an aminoacid substitution with respect to a genotype A or D reference sequence. Results: At least one immune-associated escape mutation was detected in 22.1% of patients with rising temporal-trend. By multivariable-analysis, genotype-D correlated with higher selection of ≥ 1 immune-associated escape mutation (OR[95%CI]:2.20[1.32-3.67], P = 0.002). In genotype-D, the presence of ≥ 1 immune-associated escape mutations was significantly higher in drug-exposed patients with drug-resistant strains than with wild-type virus (29.5% vs 20.3% P = 0.012). Result confirmed by ana

Cellular HIV-1 DNA Levels in Drug Sensitive Strains Are Equivalent to Those in Drug Resistant Strains in Newly- Diagnosed Patients in Europe

Background: HIV-1 genotypic drug resistance is an important threat to the success of antiretroviral therapy and transmitted resistance has reached 9 % prevalence in Europe. Studies have demonstrated that HIV-1 DNA load in peripheral blood mononuclear cells (PBMC) have a predictive value for disease progression, independently of CD4 counts and plasma viral load. Methodology/Principal Findings: Molecular-beacon-based real-time PCR was used to measure HIV-1 second template switch (STS) DNA in PBMC in newly-diagnosed HIV-1 patients across Europe. These patients were representative for the HIV-

Cellular HIV-1 DNA Levels in Drug Sensitive Strains Are Equivalent to Those in Drug Resistant Strains in Newly-Diagnosed Patients in Europe

Background: HIV-1 genotypic drug resistance is an important threat to the success of antiretroviral therapy and transmitted resistance has reached 9% prevalence in Europe. Studies have demonstrated that HIV-1 DNA load in peripheral blood mononuclear cells (PBMC) have a predictive value for disease progression, independently of CD4 counts and plasma viral load. Methodology/Principal Findings: Molecular-beacon-based real-time PCR was used to measure HIV-1 second template switch (STS) DNA in PBMC in newly-diagnosed HIV-1 patients across Europe. These patients were representative for the HIV-1 epidemic in the participating countries and were carrying either drug-resistant or sensitive viral strains. The assay design was improved from a previous version to specifically detect M-group HIV-1 and human CCR5 alleles. The findings resulted in a median of 3.32 log(10) HIV-1 copies/10(6) PBMC and demonstrated for the first time no correlation between cellular HIV-1 DNA load and transmitted drug-resistance. A weak association between cellular HIV-1 DNA levels with plasma viral RNA load and CD4(+) T-cell counts was also reconfirmed. Co-receptor tropism for 91% of samples, whether or not they conferred resistance, was CCR5. A comparison of pol sequences derived from RNA and DNA, resulted in a high similarity between the two. Conclusions/Significance: An improved molecular-beacon-based real-time PCR assay is reported for the measurement of HIV-1 DNA in PBMC and has investigated the association between cellular HIV-1 DNA levels and transmitted resistance to antiretroviral therapy in newly-diagnosed patients from across Europe. The findings show no correlation between these two parameters, suggesting that transmitted resistance does not impact disease progression in HIV-1 infected individuals. The CCR5 co-receptor tropism predominance implies that both resistant and non-resistant strains behave similarly in early infection. Furthermore, a correlation found between RNA-and DNA-derived sequences in the pol region suggests that genotypic drug-resistance testing could be carried out on either template

Characteristics of study subjects.

<p>^<i>a</i>IQR, interquartile range.</p><p>^<i>b</i>Data available for 130 patients.</p

Thermal denaturation curves of the molecular beacons and standard curves for quantification.

<p>On the left are normalised fluorescence thermal transitions of molecular beacon (pink circles) and beacon-target complexes (blue squares) designed for the detection of the human <i>CCR5</i> gene and HIV-1 STS DNA. Fluorescence signals differ between the complementary molecular-beacon-target hybrids and the non-hybridized molecular beacon at temperatures below 60°C. At higher temperatures, secondary structures within and between oligonucleotides are denatured and the beacon is free in solution in a dynamic open conformation. The temperature selected (55°C) for hybridisation in the standard PCR reactions allows optimal resolution of the fluorescence signal. On the right are standard curves for human CCR5 and HIV-1 STS DNA templates used in the real-time PCR assays for quantifying HIV-1 STS DNA in human PBMC. Six serial dilutions ranging from 10⁶ to 10¹ DNA templates were made for each DNA standard, and all standard dilutions were measured by real-time PCR using nucleotide sequence-specific molecular beacons. Median C_T values (± standard deviations) were measured for a number of replicates for each dilution point, indicated on the standard curves. The correlation coefficients (R²) of the standard curves were >0.99, and the PCR efficiencies were >99%.</p

Cellular HIV-1 DNA load in drug-resistant and non-resistant samples.

<p>Dot plot of log₁₀ cellular HIV-1 STS DNA load per million PBMC among 161 newly-diagnosed individuals with major drug-resistant mutations (n = 19) and with no major drug-resistant mutations (n = 142). The results indicate a trend towards higher cellular HIV-1 STS DNA load (<i>P</i> = 0.14) in resistant samples (median 3.64, IQR 2.63–4.25) compared to non-resistant samples (median 3.27, IQR 2.90–3.73).</p

Molecular beacons and primers used in the real-time PCR assay.

<p>^<i>a</i>FAM, fluorescein; TET, Tetrachloro-6′-carbofluorescein; Dabcyl, 4-(4′-dimethylamino phenylazo)benzoic acid; underlined sequences indicate the complementary sequences forming the molecular beacon hairpin structures.</p><p>^<i>b</i>Positions correspond to the GenBank sequences K03455 and U83326.1 for HIV-1 and CCR5, respectively. For molecular beacons, the nucleotide positions correspond to the target recognition sequences (non-underlined sequences).</p

Schematic diagram of the HIV-1 assay design.

<p>Schematic diagram of the HIV-1 genome (A) and the region targeted in this study (B). Below (C), a sequence alignment of the HXB2 strain and consensus sequences of the most widely distributed subtypes in the M group from nucleotide position 600 to 800 according to the numbering of strain HXB2, constructed from sequences available on the Los Alamos HIV sequence database (labelled as CONS). For each of the subtypes H, CRF03_AB and CRF08_BC only one sequence was available in this region and a consensus could not be made (labelled as STRAIN). No sequences were available for subtypes J and K in this region. The sequences and names of the primers and molecular beacon used in this study are seen in bold above the alignment. Directly below the beacon and reverse primer sequence is the complementary sequence corresponding to the viral positive strand. Above the beacon sequence is a schematic representation of the beacon in its closed conformation (hairpin loop). The beacon probe is labelled with a fluorophore (FAM) on the 5′end (seen as a green circle) and a DABCYL quencher on the 3′end (seen as a dark grey circle). The primer and molecular beacon sequences and their exact targets in the alignments are highlighted in grey. Dots in the sequences represent unseen regions of the alignment and dashes represent gaps in the sequences produced by the alignment.</p