Epstein Barr viral load monitoring by quantitative PCR in renal transplant patients

Post-transplant lymphoproliferative disorders (PTLD), ranging from lymphoid hyperplasia to clonal malignancy, are a severe complication arising in solid organ transplant patients. Their reported incidence ranges from 1 to 20%, according to factors such as type of transplanted organ and the age of recipients. A strong correlation between Epstein Barr virus (EBV) infection, the grade and type of immunosuppression and the development of PTLD has been recognized. The detection and quantification of EBV-DNA load in peripheral blood have been utilized as prognostic markers for the development of PTLD, showing a correlation between high levels of EBV-DNA in the blood and the development of PTLD. In this study, we monitored EBV viral load monthly in 15 renal transplant recipients for six months. The number of EBV-DNA copies was measured in peripheral blood mononuclear cells (PBMC) and serum samples by a quantitative PCR protocol developed in our laboratory that employes a previous screening of samples containing a significant number of viral DNA copies (> or =1000 copies/10(5) PBMC or 100 microl serum) by semi-quantitative PCR followed by a precise quantification of the only significant samples by quantitative-competitive (QC)-PCR. Our 15 renal transplant patients neither developed PTLD nor had recurrent acute illnesses or acute graft rejections during the study. The results obtained in the monthly follow up of EB viral load in PBMC samples confirmed its fluctuation in asymptomatic patients reported in literature. In particular, 5/14 (35.7%) of EBV seropositive patients had an EBV-DNA load equal to 1000 EBV copies /10(5) PBMC (roughly corresponding to 10.000 copies/microg PBMC DNA), and 1/14 (7.1%) reached 5000 EBV copies /10(5) PBMC (roughly corresponding to 50.000 copies/microg PBMC DNA), at least once in our study. In the EBV seronegative patient, EBV-DNA in PBMC samples was always undetectable (less than 100 DNA copies/10(5) PBMC). EBV-DNA load in all serum samples was less than threshold value of our quantification protocol (<100 DNA copies/100 microl serum), supporting the literature data. With regard to immunosuppressive treatment, 66.7% of the six patients in whom EBV load reached values equal to or higher than 1000 DNA copies/10(5) PBMC, were on FK506 whereas only 33.3% of them were on CyA. In conclusion, further investigations are needed to better understand the role of EBV infection in the pathogenesis of PTLD in immunosuppressed patients. Given the high positive predictive value of EB viral load in peripheral blood for diagnosis of PTLD reported by several authors, and the described absence of correlation between the serological evidence of EBV reactivation and EB viral load, EBV viral load measurement in PBMC and serum samples using quantitative PCR techniques is a powerful diagnostic tool to monitor transplanted patients at risk to develop PTLD.     

Mutagenesis-mediated virus extinction: virus-dependent effect of viral load on sensitivity to lethal defection

Background

Lethal mutagenesis is a transition towards virus extinction mediated by enhanced mutation rates during viral genome replication, and it is currently under investigation as a potential new antiviral strategy. Viral load and virus fitness are known to influence virus extinction. Here we examine the effect or the multiplicity of infection (MOI) on progeny production of several RNA viruses under enhanced mutagenesis.

Results

The effect of the mutagenic base analogue 5-fluorouracil (FU) on the replication of the arenavirus lymphocytic choriomeningitis virus (LCMV) can result either in inhibition of progeny production and virus extinction in infections carried out at low multiplicity of infection (MOI), or in a moderate titer decrease without extinction at high MOI. The effect of the MOI is similar for LCMV and vesicular stomatitis virus (VSV), but minimal or absent for the picornaviruses foot-and-mouth disease virus (FMDV) and encephalomyocarditis virus (EMCV). The increase in mutation frequency and Shannon entropy (mutant spectrum complexity) as a result of virus passage in the presence of FU was more accentuated at low MOI for LCMV and VSV, and at high MOI for FMDV and EMCV. We present an extension of the lethal defection model that agrees with the experimental results.

Conclusions

(i) Low infecting load favoured the extinction of negative strand viruses, LCMV or VSV, with an increase of mutant spectrum complexity. (ii) This behaviour is not observed in RNA positive strand viruses, FMDV or EMCV. (iii) The accumulation of defector genomes may underlie the MOI-dependent behaviour. (iv) LCMV coinfections are allowed but superinfection is strongly restricted in BHK-21 cells. (v) The dissimilar effects of the MOI on the efficiency of mutagenic-based extinction of different RNA viruses can have implications for the design of antiviral protocols based on lethal mutagenesis, presently under development.     

Sampling variability between two mid-turbinate swabs of the same patient has implications for influenza viral load monitoring

Background

With the clinical development of several antiviral intervention strategies for influenza, it becomes crucial to explore viral load shedding in the nasal cavity as a biomarker for treatment success, but also to explore sampling strategies for sensible and reliable virus collection.

Findings

In this study, 244 patients suffering from Influenza like Illness and/or acute respiratory tract infection were enrolled. Sampling was done using mid-turbinate flocked swabs and two swabs per patient were collected (one swab per nostril). The influenza A viral loads of two mid-turbinate flocked swabs (one for each nostril) per patient were compared and we have also assessed whether normalization for human cellular DNA in the swabs could be useful. The Influenza mid-turbinate nasal swab testing resulted in considerable sampling variability that could not be normalized against co-isolated human cellular DNA.

Conclusions

Influenza viral load monitoring in nasal swabs could be very valuable as virological endpoints in clinical trials to monitor treatment efficacy, in analogy to HIV, HBV & HCV viral load monitoring. However, the differences between left and right nostrils, as observed in this study, highlight the importance of proper sampling and the need for standardized sampling procedures.

     

Stochastic model of autocrine and paracrine signals in cell culture assays

Autocrine signaling systems are commonly studied under cell culture conditions. In a typical cell culture assay, a layer of liquid medium covers a random two-dimensional dispersion of cells, which secrete ligands. In a growing number of experiments, it is important to characterize the spatial range of autocrine and paracrine cell communication. Currently, the spatial distribution of diffusing signals can be analyzed only indirectly, from their effects on the intracellular signaling or physiological responses of autocrine cells. To directly characterize the spatial range of secreted ligands, we propose a stochastic model for autocrine cell cultures and analyze it using a combination of analytical and computational tools. The two main results derived within the framework of this model are 1), an expression for the fraction of autocrine trajectories, i.e., the probability for a ligand to be trapped by the same cell from which it has been secreted; and 2), an expression for the spatial distribution of trapping points of paracrine trajectories. We test these analytical results by stochastic simulations with efficient Brownian dynamics code and apply our model to analyze the spatial operation of autocrine epidermal growth factor receptor systems.     

Predicting the sensitivity and specificity of published real-time PCR assays

Background

In recent years real-time PCR has become a leading technique for nucleic acid detection and quantification. These assays have the potential to greatly enhance efficiency in the clinical laboratory. Choice of primer and probe sequences is critical for accurate diagnosis in the clinic, yet current primer/probe signature design strategies are limited, and signature evaluation methods are lacking.

Methods

We assessed the quality of a signature by predicting the number of true positive, false positive and false negative hits against all available public sequence data. We found real-time PCR signatures described in recent literature and used a BLAST search based approach to collect all hits to the primer-probe combinations that should be amplified by real-time PCR chemistry. We then compared our hits with the sequences in the NCBI taxonomy tree that the signature was designed to detect.

Results

We found that many published signatures have high specificity (almost no false positives) but low sensitivity (high false negative rate). Where high sensitivity is needed, we offer a revised methodology for signature design which may designate that multiple signatures are required to detect all sequenced strains. We use this methodology to produce new signatures that are predicted to have higher sensitivity and specificity.

Conclusion

We show that current methods for real-time PCR assay design have unacceptably low sensitivities for most clinical applications. Additionally, as new sequence data becomes available, old assays must be reassessed and redesigned. A standard protocol for both generating and assessing the quality of these assays is therefore of great value. Real-time PCR has the capacity to greatly improve clinical diagnostics. The improved assay design and evaluation methods presented herein will expedite adoption of this technique in the clinical lab.     

Mutation load and mutation-selection-balance in quantitative genetic traits

Haldane (1937) showed that the reduction of equilibrium mean fitness in an infinite population due to recurrent deleterious mutations depends only on the mutation rate but not on the harmfulness of mutants. His analysis, as well as more recent ones (cf. Crow 1970), ignored back mutation. The purpose of the present paper is to extend these results to arbitrary mutation patterns among alleles and to quantitative genetic traits. We derive first-order approximations for the equilibrium mean fitness (and the mutation load) and determine the order of the error term. For a metric trait under mutation-stabilizing-selection balance our result differs qualitatively from that of Crow and Kimura (1964), whose analysis is based on a Gaussian assumption. Our general approach also yields a mathematical proof that the variance under the usual mutation-stabilizing-selection model is, to first order, µ/s (the house-of-cards approximation) as µ/s tends to zero. This holds for arbitrary mutant distributions and does not require that the population mean coincide with the optimum. We show how the mutant distribution determines the order of the error term, and thus the accuracy of the house-of-cards approximation. Upper and lower bounds to the equilibrium variance are derived that deviate only to second order as µ/s tends to zero. The multilocus case is treated under the assumption of global linkage equilibrium.     

Stochastic dynamics and a power law for measles variability.

Since the discovery of a power law scaling between the mean and variance of natural populations, this phenomenon has been observed for a variety of species. Here, we show that the same form of power law scaling also occurs in measles case reports in England and Wales. Remarkably this power law holds over four orders of magnitude. We consider how the natural experiment of vaccination affects the slope of the power law. By examining simple generic models, we are able to predict the effects of stochasticity and coupling and we propose a new phenomenon associated with the critical community size.     

Stochastic model of template-directed elongation processes in biology

We present a novel modular, stochastic model for biological template-based linear chain elongation processes. In this model, elongation complexes (ECs; DNA polymerase, RNA polymerase, or ribosomes associated with nascent chains) that span a finite number of template units step along the template, one after another, with semaphore constructs preventing overtaking. The central elongation module is readily extended with modules that represent initiation and termination processes. The model was used to explore the effect of EC span on motor velocity and dispersion, and the effect of initiation activator and repressor binding kinetics on the overall elongation dynamics. The results demonstrate that (1) motors that move smoothly are able to travel at a greater velocity and closer together than motors that move more erratically, and (2) the rate at which completed chains are released is proportional to the occupancy or vacancy of activator or repressor binding sites only when initiation or activator/repressor dissociation is slow in comparison with elongation.     

Stochastic and deterministic processes jointly structure tropical arthropod communities

The question of whether ecological assemblages are structured by stochastic and deterministic (e.g. interspecific competition) processes is controversial, but it is difficult to design sampling regimes and experiments that can dissect the relative importance of stochastic and deterministic processes in natural assemblages. Using null models, we tested communities of arthropod decomposers in tropical epiphytes for patterns of species co-occurrence, while controlling for habitat gradients, seasonal variations and ecological succession. When environmental conditions were controlled, our analysis showed that the communities were structured stochastically. However, analysing mixed sets of communities that were deliberately created either from two distinct heights or two successional stages revealed that communities were structured deterministically. These results confirm that habitat gradients and dispersal/competition trade-offs are capable of generating non-random patterns within decomposer arthropod communities, but reveal that when such effects are accounted for, species co-occurrence is fundamentally random.     

Quantitative evaluation of sensitivity and selectivity of multiplex nanoSPR biosensor assays

A new functionalization procedure was developed to replace cyltrimethylammoniumbromide coating on gold nanorods (GNRs) fabricated through seed-mediated growth with chemically active alkanethiols; antibodies were then attached to the GNRs to yield gold nanorod molecular probes (GNrMPs). The functionalization procedure was shown to minimize nonspecific binding. Multiplex sensing was demonstrated for three targets (goat anti-human IgG, goat anti-rabbit IgG, and goat anti-mouse IgG) through the distinct response of the plasmon spectra of GNrMPs to binding events. Quantification of the plasmonic binding events and estimation of ligand binding kinetics tethered to these nanoscale structures was also demonstrated through a mathematical approach. Evaluation of the experimental and theoretical data yields an affinity constant K(a) = 1.34 x 10(7) M(-1), which was in agreement with the IgG-antiIgG binding affinity reported in the literature. The GNrMP sensors were found to be highly specific and sensitive with the dynamic response in the range between 10(-9) M and 10(-6) M. The limit of detection of GNrMPs was found to be in the low nanomolar range, and is a function of the binding affinity: for a higher probe-target affinity pair, the limit of detection can be expected to reach femto molar levels. This technique can play a key role in developing tunable sensors for sensitive and precise monitoring of biological interactions.     

Stochastic relations between species richness and the variability of species composition

Ecological communities change over time and space, and ecologists have long suggested that biodiversity might influence the rate of change. Here we cast new light on this question by demonstrating statistical covariation between species richness and the variability of species abundances and identities within a community (compositional variability). We provide a new analytical framework for several previously published measures of ecosystem functioning and compositional variability. We derive three related variances, each of which measures compositional variability due solely to stochastic sampling processes. Our analyses show that whether relations between species richness and compositional variability are positive, negative or zero, depends on two factors. Not only does the particular variance used affect the relation, but, more importantly, the underlying determinants of species richness can strongly affect the stochastic relations between species richness and compositional variability. This analysis makes clear for the first time how species richness should correlate with important measures of community variability, even in the absence of systematic processes.