Comparison of Droplet Digital PCR and Seminested Real-Time PCR for Quantification of Cell-Associated HIV-1 RNA

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 (N = 34) were quantified with both methods. Correlations were observed between the methods both for serially diluted synthetic standards (usRNA: R² = 0.97, msRNA: R² = 0.92) and patient-derived samples (usRNA: R² = 0.51, msRNA: R² = 0.87). 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 (p = 0.13). We observed an average agreement between the methods for usRNA quantification in patient samples, albeit with a large standard deviation (bias = 0.05±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.     

Quantification of unintegrated HIV-1 DNA at the single cell level in vivo

In the nucleus of HIV-1 infected cells, unintegrated HIV-1 DNA molecules exist in the form of one and two LTR circles and linear molecules with degraded extremities. In tissue culture they are invariably more numerous than the provirus, the relative proportion of integrated to unintegrated forms varies widely from ～1:1 to 1:10 and even over 1:100. In vivo, this ratio is unknown. To determine it, single nuclei from two infected patients with a known provirus copy number were microdissected, HIV DNA was amplified by nested PCR, cloned and individual clones sequenced. Given the extraordinary sequence complexity, we made the assumption that the total number of distinct sequences approximated to real number of amplifiable HIV-1 DNA templates in the nucleus. We found that the number of unintegrated DNA molecules increased linearly with the proviral copy number there being on average 86 unintegrated molecules per provirus.     

A Comparison of Colorimetric and DNA Quantification Assays for the Assessment of Meniscal Fibrochondrocyte Proliferation in Microcarrier Culture

We have developed and refined a rapid, reliable method for the evaluation of attachment and proliferation of ovine meniscal chondrocytes in microcarrier culture. Assays measuring both mitochondrial activity, using MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide] and MTS [3-(4,5-dimethylthiazol-2-yl)5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium], and DNA synthesis with a PicoGreen assay were compared. The MTT assay was the most sensitive at lower cell concentrations and enabled accurate assessment of cell proliferation over 14 day culture.     

Real-Time PCR Assays for the Quantification of HCV RNA: Concordance,Discrepancies and Implications for Response Guided Therapy

Background and Aims

Monitoring of chronic Hepatitis C (CHC) treatment relies on HCV RNA quantification by means of real-time PCR methods. Assay specific analytical sensitivities may impact therapy management.

Methods

Comparative analysis between three commercial assays (Roche COBAS AmpliPrep/COBAS TaqMan Version 1 (CAP/CTM Ver. 1), Version 2 (CAP/CTM Ver. 2) and the Abbott RealTime HCV (ART) assay) was performed on 247 available samples taken at key decision time points during antiviral therapy of 105 genotype 1 patients (triple therapy: n = 70; dual therapy: n = 35).

Results

Overall concordance of HCV RNA measurements was high between the two Roche systems (89%; n = 220/247) but lower between the Roche assays and the ART (CAP/CTM Ver. 1 vs ART: 77.3%; n = 191/247 and CAP/CTM v.2 vs ART: 80.1%; n = 198/247). Most discrepancies were noted in week 4/8 samples with residual viremia (<LLOQ) detected by ART (<LLOQ: n = 45, 44.1%) but undetectable HCV RNA by CAP/CTM Ver. 1 (<LLOQ: n = 18, 17.6%) or CAP/CTM Ver. 2 (<LLOQ: n = 26, 25.5%). Based on results by CAP/CTM Ver. 1, 13 eligible patients underwent an abbreviated course of therapy (24 weeks). Only 1 patient experienced virologic breakthrough. If tested by ART, only 6/13 patients (46.2%) would have been eligible for shortened treatment. Consequently, RGT guidelines were adapted and shortening of therapy was allowed if residual viremia was detected by ART at week 4/8.

Conclusion

An abbreviated course of treatment can safely be applied in patients with residual viremia (<LLOQ) detected by ART in samples collected at week 4/8 of treatment.     

Base Pairing at the Stem-loop Junction in the SL1 Kissing Complex of HIV-1 RNA: A Thermodynamic Study Probed by Molecular Dynamics Simulation

Abstract

The thermodynamics of the opening/closure process of a GC base pair located at the stem-loop junction of the SL1 sequence from HIV-1_Lai genomic RNA was investigated in the context of a loop-loop homodimer (or kissing complex) using molecular dynamics simulation. The free energy, enthalpy and entropy changes for the closing reaction are 0 kcal·mol^?1, ?11 kcal·mol^?1and ?0.037 kcal·mol^?1-K^?1 at 300° K respectively. Furthermore it is found that the free energy change is the same for the formation of a 11 nucleotide loop closed with UG and for the formation of a 9 nucleotide loop closed with GC. Our study evidences the high flexibility of the nucleotides at the stem-loop junction explaining the weak stability of this structure.     

Thermodynamic examination of trinucleotide bulged RNA in the context of HIV-1 TAR RNA

RNA structures contain many bulges and loops that are expected to be sites for inter- and intra-molecular interactions. Nucleotides in the bulge are expected to influence the structure and recognition of RNA. The same stability is assigned to all trinucleotide bulged RNA in the current secondary structure prediction models. In this study thermal denaturation experiments were performed on four trinucleotide bulged RNA, in the context of HIV-1 TAR RNA, to determine whether the bulge sequence affects RNA stability and its divalent ion interactions. Cytosine-rich bulged RNA were more stable than uracil-rich bulged RNA in 1 M KCl. Interactions of divalent ions were more favorable with uracil-rich bulged RNA by ~2 kcal/mol over cytosine-rich bulged RNA. The UCU-TAR RNA (wild type) is stabilized by 1.7 kcal/mol in 9.5 mM Ca²⁺ as compared with 1 M KCl, whereas no additional gain in stability is measured for CCC-TAR RNA. These results have implications for base substitution experiments traditionally employed to identify metal ion binding sites. To our knowledge, this is the first systematic study to quantify the effect of small sequence changes on RNA stability upon interactions with divalent ions.     

Development and Evaluation of PCR Assays for the Detection of Paenibacillus larvae in Honey Samples: Comparison with Isolation and Biochemical Characterization

PCR assays were developed for the direct detection of Paenibacillus larvae in honey samples and compared with isolation and biochemical characterization procedures. Different primer pairs, designed from the 16S rRNA and the metalloproteinase precursor gene regions, and different DNA extraction methods were tested and compared. The sensitivity of the reactions was evaluated by serial dilutions of DNA extracts obtained from P. larvae cultures. The specificity of the primers was assessed by analyzing related Paenibacillus and Bacillus strains isolated from honey. The PCR assays also amplified these related bacteria, but at lower sensitivity. In the next step, the PCR assays were applied to contaminated honey and other bee products originating from 15 countries. Lysozyme treatment followed by proteinase K digestion was determined to be the best DNA extraction method for P. larvae spores. The most sensitive primer pair detected P. larvae in 18 of 23 contaminated honey samples, as well as in pollen, wax, and brood. Honey specimens containing saprophyte bacilli and paenibacilli, but not P. larvae, were PCR negative. Although the isolation and biochemical identification method (BioLog) showed higher sensitivity and specificity, PCR proved to be a valuable technique for large-scale screening of honey samples for American foulbrood, especially considering its rapidity and moderate costs.     

Dimerization of HIV-1 genomic RNA of subtypes A and B: RNA loop structure and magnesium binding.

Retroviruses encapsidate their genome as a dimer of homologous RNA molecules noncovalently linked close to their 5' ends. The dimerization initiation site (DIS) of human immunodeficiency virus type 1 (HIV-1) RNA is a hairpin structure that contains in the loop a 6-nt self-complementary sequence flanked by two 5' and one 3' purines. The self-complementary sequence, as well as the flanking purines, are crucial for dimerization of HIV-1 RNA, which is mediated by formation of a "kissing-loop" complex between the DIS of each monomer. Here, we used chemical modification interference, lead-induced cleavage, and three-dimensional modeling to compare dimerization of subtype A and B HIV-1 RNAs. The DIS loop sequences of these RNAs are AGGUGCACA and AAGCGCGCA, respectively. In both RNAs, ethylation of most but not all phosphate groups in the loop and methylation of the N7 position of the G residues in the self-complementary sequence inhibited dimerization. These results demonstrate that small perturbations of the loop structure are detrimental to dimerization. Conversely, methylation of the N1 position of the first and last As in the loop were neutral or enhanced dimerization, a result consistent with these residues forming a noncanonical sheared base pair. Phosphorothioate interference, lead-induced cleavage, and Brownian-dynamics simulation revealed an unexpected difference in the dimerization mechanism of these RNAs. Unlike subtype B, subtype A requires binding of a divalent cation in the loop to promote RNA dimerization. This difference should be taken into consideration in the design of antidimerization molecules aimed at inhibiting HIV-1 replication.     

RNA interactions in the 5' region of the HIV-1 genome

The untranslated leader of the dimeric HIV-1 RNA genome is folded into a complex structure that plays multiple and essential roles in the viral replication cycle. Here, we have investigated secondary and tertiary structural elements within the 5' 744 nucleotides of the HIV-1 genome using a combination of bioinformatics, enzymatic probing, native gel electrophoresis, and UV-crosslinking experiments. We used a recently developed RNA folding algorithm (Pfold) to predict the common secondary structure of an alignment of 20 divergent HIV-1 sequences. Combining this analysis with biochemical data, we present a secondary structure model for the entire 744 nucleotide fragment, which incorporates previously recognized and novel structural elements. In particular, our data provided strong evidence for a long-distance interaction between the region encompassing the AUG Gag initiation codon and an upstream region and we demonstrate that this feature is highly conserved in distantly related human and animal retroviruses. To obtain information about tertiary interactions we applied an intramolecular UV-crosslinking strategy and identified a novel tertiary interaction within the PBS hairpin structure.     

Comparison of relative mRNA quantification models and the impact of RNA integrity in quantitative real-time RT-PCR

Relative quantification in quantitative real-time RT-PCR is increasingly used to quantify gene expression changes. In general, two different relative mRNA quantification models exist: the delta-delta Ct and the efficiency-corrected Ct model. Both models have their advantages and disadvantages in terms of simplification on the one hand and efficiency correction on the other. The particular problem of RNA integrity and its effect on relative quantification in qRT-PCR performance was tested in different bovine tissues and cell lines (n = 11). Therefore different artificial and standardized RNA degradation levels were used. Currently fully automated capillary electrophoresis systems have become the new standard in RNA quality assessment. RNA quality was rated according the RNA integrity number (RIN). Furthermore, the effect of different length of amplified products and RNA integrity on expression analyses was investigated. We found significant impact of RNA integrity on relative expression results, mainly on cycle threshold (Ct) values and a minor effect on PCR efficiency. To minimize the interference of RNA integrity on relative quantification models, we can recommend to normalize gene expression by an internal reference gene and to perform an efficiency correction. Results demonstrate that innovative new quantification methods and normalization models can improve future mRNA quantification.     

Evaluation of the NucliSens EasyQ v2.0 Assay in Comparison with the Roche Amplicor v1.5 and the Roche CAP/CTM HIV-1 Test v2.0 in Quantification of C-Clade HIV-1 in Plasma

Human immunodeficiency virus type 1 (HIV-1) genetic diversity poses a challenge to reliable viral load monitoring. Discrepancies between different testing platforms have been observed, especially for non-clade-B virus. Therefore we compare, in antiretroviral therapy (ART)-naïve South African subjects predominantly infected with HIV-1 clade-C, three commercially available assays: the COBAS AmpliPrep/COBAS TaqMan HIV-1 Test version 2.0 by Roche (CAP/CTM v2.0), the BioMérieux NucliSens Version 2.0 Easy Q/Easy Mag (NucliSens v2.0) and the Roche COBAS Amplicor HIV-1 Monitor Test Version 1.5 (Amplicor v1.5). Strong linear correlation was observed and Bland-Altman analyses showed overall good agreement between the assays with mean viral load differences of 0.078 log cp/ml (NucliSens v2.0 – Amplicor v1.5), 0.260 log cp/ml (CAP/CTM v2.0 – Amplicor v1.5) and 0.164 log cp/ml (CAP/CTM v2.0 – NucliSens v2.0), indicating lower mean viral load results for the Amplicor v1.5 and higher mean readings for the CAP/CTM v2.0. Consistent with observations following previous comparisons of CAP/CTM v2.0 versus Amplicor v1.5, the CAP/CTM v2.0 assay detected low-level viremia (median 65 cp/ml) in more than one-third of those in whom viremia had been undetectable (<20 cp/ml) in assays using the NucliSens platform. These levels of viremia are of uncertain clinical significance but may be of importance in early detection of ART resistance in those on treatment. Overall the three assays showed good comparability of results but with consistent, albeit relatively small, discrepancies for HIV-1 clade-C samples, especially in the low-viremic range that should be taken into account when interpreting viral load data.     

HIV-1 protease inhibitors: enthalpic versus entropic optimization of the binding affinity

Existing experimental as well as computational screening methods select potential ligands or drug candidates on the basis of binding affinity. Since the binding affinity is a function of the enthalpy (DeltaH) and entropy (DeltaS) changes, it is apparent that improved binding can be achieved in different ways: by optimizing DeltaH, DeltaS, or a combination of both. However, the behavior of enthalpically or entropically optimized inhibitors is fundamentally different, including their response to mutations that may elicit drug resistance. In the design of HIV-1 protease inhibitors, high binding affinity has usually been achieved by preshaping lead compounds to the geometry of the binding site and by incorporating a high degree of hydrophobicity. The thermodynamic consequence of that approach is that the binding affinity of the resulting inhibitors becomes entropically favorable but enthalpically unfavorable. Specifically, the resulting high binding affinity is due to an increased solvation entropy (hydrophobic effect) combined with a reduced loss of conformational entropy of the inhibitor upon binding (structural rigidity). Here we report that tripeptide inhibitors derived from the transframe region of Gag-Pol (Glu-Asp-Leu and Glu-Asp-Phe) bind to the HIV-1 protease with a favorable enthalpy change. This behavior is qualitatively different from that of known inhibitors and points to new strategies for inhibitor design. Since the binding affinities of enthalpically favorable and enthalpically unfavorable inhibitors have opposite temperature dependence, it is possible to design fast screening protocols that simultaneously select inhibitors on the basis of affinity and enthalpy.