A cost effective real-time PCR for the detection of adenovirus from viral swabs

Compared to traditional testing strategies, nucleic acid amplification tests such as real-time PCR offer many advantages for the detection of human adenoviruses. However, commercial assays are expensive and cost prohibitive for many clinical laboratories. To overcome fiscal challenges, a cost effective strategy was developed using a combination of homogenization and heat treatment with an “in-house” real-time PCR. In 196 swabs submitted for adenovirus detection, this crude extraction method showed performance characteristics equivalent to viral DNA obtained from a commercial nucleic acid extraction. In addition, the in-house real-time PCR outperformed traditional testing strategies using virus culture, with sensitivities of 100% and 69.2%, respectively. Overall, the combination of homogenization and heat treatment with a sensitive in-house real-time PCR provides accurate results at a cost comparable to viral culture

Predicting the Next Eye Pathogen: Analysis of a Novel Adenovirus

For DNA viruses, genetic recombination, addition, and deletion represent important evolutionary mechanisms. Since these genetic alterations can lead to new, possibly severe pathogens, we applied a systems biology approach to study the pathogenicity of a novel human adenovirus with a naturally occurring deletion of the canonical penton base Arg-Gly-Asp (RGD) loop, thought to be critical to cellular entry by adenoviruses. Bioinformatic analysis revealed a new highly recombinant species D human adenovirus (HAdV-D60). A synthesis of in silico and laboratory approaches revealed a potential ocular tropism for the new virus. In vivo, inflammation induced by the virus was dramatically greater than that by adenovirus type 37, a major eye pathogen, possibly due to a novel alternate ligand, Tyr-Gly-Asp (YGD), on the penton base protein. The combination of bioinformatics and laboratory simulation may have important applications in the prediction of tissue tropism for newly discovered and emerging viruses

Influence of Novel Norovirus GII.4 Variants on Gastroenteritis Outbreak Dynamics in Alberta and the Northern Territories, Canada between 2000 and 2008

BACKGROUND: Norovirus GII.4 is the predominant genotype circulating worldwide over the last decade causing 80% of all norovirus outbreaks with new GII.4 variants reported in parallel with periodic epidemic waves of norovirus outbreaks. The circulating new GII.4 variants and the epidemiology of norovirus outbreaks in Alberta, Canada have not been described. Our hypothesis is that the periodic epidemic norovirus outbreak activity in Alberta was driven by new GII.4 variants evolving by genetic drift. METHODOLOGY/PRINCIPAL FINDINGS: The Alberta Provincial Public Health Laboratory performed norovirus testing using RT-PCR for suspected norovirus outbreaks in the province and the northern Territories between 2000 and 2008. At least one norovirus strain from 707 out of 1,057 (66.9%) confirmed norovirus outbreaks were successfully sequenced. Phylogenetic analysis was performed using BioNumerics and 617 (91.1%) outbreaks were characterized as caused by GII.4 with 598 assigned as novel variants including: GII.4-1996, GII.4-2002, GII.4-2004, GII.4-2006a, GII.4-2006b, GII.4-2008a and GII.4-2008b. Defining July to June of the following year as the yearly observation period, there was clear biannual pattern of low and high outbreak activity in Alberta. Within this biannual pattern, high outbreak activity followed the emergence of novel GII.4 variants. The two variants that emerged in 2006 had wider geographic distribution and resulted in higher outbreak activity compared to other variants. The outbreak settings were analyzed. Community-based group residence was the most common for both GII.4 variants and non-GII.4 variants. GII.4 variants were more commonly associated with outbreaks in acute care hospitals while outbreaks associated non-GII.4 variants were more commonly seen in school and community social events settings (p<0.01). CONCLUSIONS/SIGNIFICANCE: The emergence of new norovirus GII.4 variants resulted in an increased norovirus outbreak activity in the following season in a unique biannual pattern in Alberta over an eight year period. The association between antigenic drift of GII.4 strains and epidemic norovirus outbreak activity could be due to changes in host immunity, viral receptor binding efficiency or virulence factors in the new variants. Early detection of novel GII.4 variants provides vital information that could be used to forecast the norovirus outbreak burden, enhance public health preparedness and allocate appropriate resources for outbreak management

Adamantane resistance in seasonal human influenza A viruses from Calgary, Alberta (January 2007 to August 2008)

The available antivirals for the treatment and prophylaxis of influenza A infections include the adamantanes (amantadine and rimantadine), which are matrix (M2) protein inhibitors, and the neuraminidase inhibitors (oseltamivir and zanamivir). Resistance to the adamantanes is conferred by mutations at amino acid positions 26, 27, 30, 31 or 34 within the M2 protein of influenza A viruses. A significant increase in adamantane resistance has been reported worldwide since 2003, reflected by a similar increase in Canada. The present study reports on the frequency of adamantane resistance in seasonal influenza A viruses in Calgary, Alberta, for the period between January 2007 and August 2008, as an update to the previous report. Positive influenza A samples (221 original patient specimens and 34 isolates obtained by viral culture) were analyzed for changes in the critical amino acid residues of the M2 gene. The amplification and sequencing of regions that confer adamantane resistance directly from RNA extracts of clinical samples (without previous culture) makes this approach a fast and efficient process for monitoring resistance. The results showed that the frequency of resistance varied from 37.5% to 49.2% in circulating influenza A H3N2 virus strains (n=213) between January 2007 and April 2007. The frequency of resistance increased to 100% in May 2007, after which all H3N2 viruses were resistant until the end of the monitoring period. All resistant H3N2 viruses contained the serine to asparagine substitution at amino acid position 31. Resistance was not observed in the H1N1 viruses tested (n=39) within this monitoring period. The level of adamantane resistance in H3N2 viruses continues to remain high since resistant viruses became the prevalent circulating strains in 2005. Recent reports have indicated that the currently circulating swine-origin influenza A H1N1 subtype viruses are adamantane resistant. It is, thus, important to continue to monitor seasonal influenza A viruses for antiviral resistance markers to ensure optimal prophylaxis and treatment

Adamantane Resistance in Seasonal Human Influenza A Viruses from Calgary, Alberta (January 2007 to August 2008)

The available antivirals for the treatment and prophylaxis of influenza A infections include the adamantanes (amantadine and rimantadine), which are matrix (M2) protein inhibitors, and the neuraminidase inhibitors (oseltamivir and zanamivir). Resistance to the adamantanes is conferred by mutations at amino acid positions 26, 27, 30, 31 or 34 within the M2 protein of influenza A viruses. A significant increase in adamantane resistance has been reported worldwide since 2003, reflected by a similar increase in Canada. The present study reports on the frequency of adamantane resistance in seasonal influenza A viruses in Calgary, Alberta, for the period between January 2007 and August 2008, as an update to the previous report. Positive influenza A samples (221 original patient specimens and 34 isolates obtained by viral culture) were analyzed for changes in the critical amino acid residues of the M2 gene. The amplification and sequencing of regions that confer adamantane resistance directly from RNA extracts of clinical samples (without previous culture) makes this approach a fast and efficient process for monitoring resistance. The results showed that the frequency of resistance varied from 37.5% to 49.2% in circulating influenza A H3N2 virus strains (n=213) between January 2007 and April 2007. The frequency of resistance increased to 100% in May 2007, after which all H3N2 viruses were resistant until the end of the monitoring period. All resistant H3N2 viruses contained the serine to asparagine substitution at amino acid position 31. Resistance was not observed in the H1N1 viruses tested (n=39) within this monitoring period. The level of adamantane resistance in H3N2 viruses continues to remain high since resistant viruses became the prevalent circulating strains in 2005. Recent reports have indicated that the currently circulating swine-origin influenza A H1N1 subtype viruses are adamantane resistant. It is, thus, important to continue to monitor seasonal influenza A viruses for antiviral resistance markers to ensure optimal prophylaxis and treatment.Peer Reviewe

Enhanced Viral Etiological Diagnosis of Respiratory System Infection Outbreaks by Use of a Multitarget Nucleic Acid Amplification Assay ▿

A study was undertaken to assess the utility of the xTAG respiratory viral panel (RVP) for enhanced laboratory investigation of respiratory outbreaks. Specimens (n = 1,108) from 244 suspected respiratory virus outbreaks in 2006 and 2007 in Alberta, Canada, were included in the study. Testing by direct fluorescent antigen detection (DFA) and various in-house nucleic acid amplification tests (NATs) for common respiratory viruses provided an etiological diagnosis in 177 outbreaks (72.5%), with 524 samples testing positive (47.3%) for a respiratory virus. Two hundred samples from 51 unresolved outbreaks were further tested by RVP retrospectively. Fifty-eight samples from 30 unresolved outbreaks had a respiratory virus detected by RVP (47 picornavirus-positive, 9 coronavirus-positive, and 2 influenza virus A-positive samples). Overall, detection of a viral etiological agent was achieved in 90.8% of outbreaks using a combination of DFA, NATs, and RVP. Use of RVP enhances the laboratory investigation of respiratory virus outbreaks and facilitates appropriate patient and outbreak management

Genetic characterization of a Coxsackie A9 virus associated with aseptic meningitis in Alberta, Canada in 2010

Article deposited according to agreement with BMC, December 2, 2010 and according to publisher policies: http://www.biomedcentral.com/about/copyright [May 29, 2013].YesFunding provided by the Open Access Authors Fund

Multiplex, Quantitative, Reverse Transcription PCR Detection of Influenza Viruses Using Droplet Microfluidic Technology

Quantitative, reverse transcription, polymerase chain reaction (qRT-PCR) is facilitated by leveraging droplet microfluidic (DMF) system, which due to its precision dispensing and sample handling capabilities at microliter and lower volumes has emerged as a popular method for miniaturization of the PCR platform. This work substantially improves and extends the functional capabilities of our previously demonstrated single qRT-PCR micro-chip, which utilized a combination of electrostatic and electrowetting droplet actuation. In the reported work we illustrate a spatially multiplexed micro-device that is capable of conducting up to eight parallel, real-time PCR reactions per usage, with adjustable control on the PCR thermal cycling parameters (both process time and temperature set-points). This micro-device has been utilized to detect and quantify the presence of two clinically relevant respiratory viruses, Influenza A and Influenza B, in human samples (nasopharyngeal swabs, throat swabs). The device performed accurate detection and quantification of the two respiratory viruses, over several orders of RNA copy counts, in unknown (blind) panels of extracted patient samples with acceptably high PCR efficiency (&gt;94%). The multi-stage qRT-PCR assays on eight panel patient samples were accomplished within 35–40 min, with a detection limit for the target Influenza virus RNAs estimated to be less than 10 RNA copies per reaction

Comparison of the Luminex xTAG Respiratory Viral Panel with In-House Nucleic Acid Amplification Tests for Diagnosis of Respiratory Virus Infections ▿

Detection of respiratory viruses using sensitive real-time nucleic acid amplification tests (NATs) is invaluable for patient and outbreak management. However, the wide range of potential respiratory virus pathogens makes testing using individual real-time NATs expensive and laborious. The objective of this study was to compare the detection of respiratory virus targets using the Luminex xTAG respiratory viral panel (RVP) assay with individual real-time NATs used at the Provincial Laboratory of Public Health, Calgary, Alberta, Canada. The study included 1,530 specimens submitted for diagnosis of respiratory infections from December 2006 to May 2007. Direct-fluorescent-antigen-positive nasopharyngeal samples were excluded from this study. A total of 690 and 643 positives were detected by RVP and in-house NATs, respectively. Kappa correlation between in-house NATs and RVP for all targets ranged from 0.721 to 1.000. The majority of specimens missed by in-house NATs (96.7%) were positive for picornaviruses. Samples missed by RVP were mainly positive for adenovirus (51.7%) or respiratory syncytial virus (27.5%) by in-house NATs and in general had low viral loads. RVP allows for multiplex detection of 20 (and differentiation between 19) respiratory virus targets with considerable time and cost savings compared with alternative NATs. Although this first version of the RVP assay has lower sensitivity than in-house NATs for detection of adenovirus, it has good sensitivity for other targets. The identification of picornaviruses and coronaviruses and concurrent typing of influenza A virus by RVP, which are not currently included in our diagnostic testing algorithm, will improve our diagnosis of respiratory tract infections