Droplet digital PCR is an accurate method to assess methylation status on FFPE samples.

Most tissue samples available for cancer research are archived as formalin-fixed paraffin-embedded (FFPE) samples. However, the fixation process and the long storage duration lead to DNA fragmentation and hinder epigenome analysis. The use of droplet digital PCR (ddPCR) to detect DNA methylation has recently emerged. In this study, we compare an optimized ddPCR assay with a conventional qPCR assay by targeting a dilution series of control DNA. In addition, we compare the ddPCR technology with results from Infinium arrays targeting two separate CpG sites on a set of colon adenoma FFPE samples. Our data demonstrate that qPCR and ddPCR assess methylation status equally well on dilution controls with a high DNA input. However, the methylation detection on low-input samples is more accurate using ddPCR. The proposed primer design (methylation-independent primers with amplification of solely the converted DNA target) will allow for methylation detection, independent of bisulfite conversion efficiency. Those data show that ddPCR can be used for methylation analysis on FFPE samples with a wide range of DNA input and that the precision of the assay depends largely on the total amount of amplifiable DNA fragments. Due to accessibility of the ddPCR technology and its accuracy on high- as well as low-DNA input samples, we propose the use of this approach for studies involving degraded FFPE samples.

Sampling and detection of airborne influenza virus towards point-of-care applications.

Airborne transmission of the influenza virus contributes significantly to the spread of this infectious pathogen, particularly over large distances when carried by aerosol droplets with long survival times. Efficient sampling of virus-loaded aerosol in combination with a low limit of detection of the collected virus could enable rapid and early detection of airborne influenza virus at the point-of-care setting. Here, we demonstrate a successful sampling and detection of airborne influenza virus using a system specifically developed for such applications. Our system consists of a custom-made electrostatic precipitation (ESP)-based bioaerosol sampler that is coupled with downstream quantitative polymerase chain reaction (qPCR) analysis. Aerosolized viruses are sampled directly into a miniaturized collector with liquid volume of 150 µL, which constitutes a simple and direct interface with subsequent biological assays. This approach reduces sample dilution by at least one order of magnitude when compared to other liquid-based aerosol bio-samplers. Performance of our ESP-based sampler was evaluated using influenza virus-loaded sub-micron aerosols generated from both cultured and clinical samples. Despite the miniaturized collection volume, we demonstrate a collection efficiency of at least 10% and sensitive detection of a minimum of 3721 RNA copies. Furthermore, we show that an improved extraction protocol can allow viral recovery of down to 303 RNA copies and a maximum sampler collection efficiency of 47%. A device with such a performance would reduce sampling times dramatically, from a few hours with current sampling methods down to a couple of minutes with our ESP-based bioaerosol sampler.

Bioassay Development for Ultrasensitive Detection of Influenza A Nucleoprotein Using Digital ELISA.

Flu is caused by the influenza virus that, due to mutations, keeps our body vulnerable for infections, making early diagnosis essential. Although immuno-based diagnostic tests are available, they have low sensitivity and reproducibility. In this paper, the prospect of detecting influenza A virus using digital ELISA has been studied. To appropriately select bioreceptors for this bioassay, seven commercial antibodies against influenza A nucleoprotein were methodically tested for their reactivity and binding affinity. The study has been performed on two markedly different platforms, being an enzyme-linked immunosorbent assay and a surface plasmon resonance system. The selected antibodies displayed completely different behavior on the two platforms and in various assay configurations. Surprisingly, the antibodies that showed overall good reactivity on both platforms had the highest dissociation constant among the tested antibodies, suggesting that, although important, binding affinity is not the only parameter to be considered when selecting antibodies. Moreover, only one antibody had the capacity to capture the nucleoprotein directly in lysis buffer used for releasing this viral protein, which might pose a huge advantage when developing assays with a fast time-to-result. This antibody was implemented on an in-house developed digital ELISA platform for ultrasensitive detection of recombinant nucleoprotein, reaching a detection limit of 4 ± 1 fM in buffer and 10 ± 2 fM in 10-fold diluted nasopharyngeal swabs, which is comparable to currently available fast molecular detection techniques. These results point to a great potential for ultrasensitive immuno-based influenza detection.

A Downward Trend of the Ratio of Influenza RNA Copy Number to Infectious Viral Titer in Hospitalized Influenza A-Infected Patients.

Background. Efficacy endpoints in influenza clinical trials may include clinical symptoms and virological measurements, although virology cannot serve as the primary endpoint. We investigated the relationship between influenza A RNA copy number and quantity of infectious viruses in hospitalized influenza patients. Methods. One hundred fifty influenza-infected, hospitalized patients were included in this prospective cohort study spanning the 2012-2013 influenza season. Daily nasopharyngeal samples were collected during hospitalization, and influenza A RNA copy number and infectious viral titer were monitored. Results. The decay rate for 50% tissue culture infectious dose (TCID50) was 0.51 ± 0.14 log10 TCID50/mL per day, whereas the RNA copy number decreased at a rate of 0.41 ± 0.04 log10 copies/mL per day (n = 433). The log ratio of the RNA copy number to the infectious viral titer within patient changes significantly with -0.25 ± 0.09 units per day (P = .0069). For a 12-day observation period, the decay corresponds to a decline of this ratio of 3 log influenza RNA copies. Conclusions. Influenza RNA copy number in nasal swabs is co-linear with culture, although the rate of decay of cell culture-based viral titers was faster than that observed with molecular methods. The study documented a clear decreasing log ratio of the RNA copy number to the infectious viral titer of the patients over time.

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.

Comparison of the FilmArray RP, Verigene RV+, and Prodesse ProFLU+/FAST+ multiplex platforms for detection of influenza viruses in clinical samples from the 2011-2012 influenza season in Belgium.

Respiratory tract infections (RTIs) are caused by a plethora of viral and bacterial pathogens. In particular, lower RTIs are a leading cause of hospitalization and mortality. Timely detection of the infecting respiratory pathogens is crucial to optimize treatment and care. In this study, three U.S. Food and Drug Administration-approved molecular multiplex platforms (Prodesse ProFLU+/FAST+, FilmArray RP, and Verigene RV+) were evaluated for influenza virus detection in 171 clinical samples collected during the Belgian 2011-2012 influenza season. Sampling was done using mid-turbinate flocked swabs, and the collected samples were stored in universal transport medium. The amount of viral RNA present in the swab samples ranged between 3.07 and 8.82 log10 copies/ml. Sixty samples were concordant influenza A virus positive, and 8 samples were found to be concordant influenza B virus positive. Other respiratory viruses that were detected included human rhinovirus/enterovirus, respiratory syncytial virus, parainfluenza virus type 1, human metapneumovirus, and coronavirus NL63. Twenty-five samples yielded discordant results across the various assays which required further characterization by sequencing. The FilmArray RP and Prodesse ProFLU+/FAST+ assays were convenient to perform with regard to sensitivity, ease of use, and low percentages of invalid results. Although the limit of sensitivity is of utmost importance, many other factors should be taken into account in selecting the most convenient molecular diagnostic assay for the detection of respiratory pathogens in clinical samples.

Evaluation of a proximity extension assay for the detection of H1 2009 pandemic influenza viruses.

The rapid influenza diagnostic tests (RIDTs) are widely distributed, simple to use, but often lack sensitivity as compared to gold standard methods (viral culture and nucleic acid detection technologies). Applying RIDTs outside of epidemic or pandemic infections results in large numbers of false negatives. Hence, a sensitive RIDT that would reduce the number of false negatives would result in an increased clinical value. We evaluated the potential of a proximity extension assay (PEA) for the detection of influenza A H1 viruses. This technology makes use of antibodies to capture the pathogen, followed by molecular detection. Forty-seven nasopharyngeal swab samples, all confirmed infections of the H1 2009 pandemic influenza virus, were evaluated. The performance of PEA was compared to the RIDT Quickvue Influenza A+B assay. The success rate of the comparative assays was modeled by means of a binary logistic response model. Both assays performed equally well within the current range of viral particles, expressed as log10 copies/ml. When the actual input of viral particles was taken into account, the 95% hitrate of PEA lies within the range of 4.60-7.02 log10 copies/reaction, which is an almost 2 log10 sensitivity improvement over the 95% hitrate of the Quickvue RIDT, ranging from 6.86 to 9.37 log10 copies/reaction. The PEA method holds promise to improve sensitive detection of influenza viruses in clinical samples.