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With the emerging Zika virus (ZIKV) epidemic, accessible real-time reverse transcription-PCR (rRT-PCR) assays are needed to streamline testing. The commercial Altona Diagnostics RealStar ZIKV rRT-PCR test kit (Altona PCR) has been approved for emergency use authorization by the U.S. FDA. Our aim was to verify the Altona PCR by comparing it to the CDC-designed dual-target ZIKV rRT-PCR reference assay (reference PCR) and describe the demographics of patients tested for ZIKV by rRT-PCR in Ontario, Canada. A large set of clinical specimens was tested for ZIKV by the Altona PCR and the reference PCR. Positive or equivocal specimens underwent PCR and Sanger sequencing targeting the ZIKV NS5 gene. A total of 671 serum specimens were tested by the reference PCR: 58 (8.6%) were positive, 193 (28.8%) were equivocal, and 420 (62.6%) were negative. Ninety percent of the reference PCR-positive patients were tested in the first 5 days after symptom onset. The Altona PCR was performed on 284/671 specimens tested by the reference PCR. The Altona PCR was positive for 53/58 (91%) reference PCR-positive specimens and 16/193 (8%) reference PCR-equivocal specimens; the ZIKV NS5 PCR was positive for all 68 Altona PCR-positive specimens and negative for all 181 Altona PCR-negative specimens that underwent the NS5 PCR. The Altona PCR has very good sensitivity (91%) and specificity (97%) compared to the reference PCR. The Altona PCR can be used for ZIKV diagnostic testing and has less extensive verification requirements than a laboratory-developed test.

Background Certain adenovirus serotypes cause severe infections, especially in children. It is important to monitor temporal changes in serotypes causing clinical disease. The objective of this study was to document circulating respiratory adenovirus serotypes by sequencing adenovirus culture isolates from the Greater Toronto Area, Ontario, during December 2008 to April 2010. Methods Nucleic acid extraction was performed on 90 respiratory tract adenovirus culture isolates. PCR amplification was conducted with primers targeting the adenovirus hexon gene hypervariable region 7. Sanger sequencing and phylogenetic analyses were performed to determine serotype identities. Results Among 90 clinical respiratory isolates sequenced, eight different serotypes were identified. Serotype 3 (34, 38%), serotype 2 (30, 30%), and serotype 1 (14, 16%) isolates were most common; serotypes 5, 6, 11, 17 and 21 were also observed. Seventeen (50%) of the 34 HAdV-3 isolates were identified between December 2008 and February 2009, while none were identified from December 2009 to February 2010. Between December 2008 and April 2009, the two most common serotypes were HAdV-3 and HAdV-2, detected in 18 (53%) and 8 (24%) of the 34 cultures isolates, respectively. However, from December 2009 to April 2010, there was an increase in HAdV-2, which became the most prevalent serotype, detected in 10 (50%) of the 20 isolates identified (p = 0.05). Conclusions There was a gradual shift in prevailing adenovirus serotypes during the 17 month study period, from predominantly HAdV-3 to HAdV-2. If an adenovirus vaccine were to be broadly implemented, multiple serotypes should be included.

Background Oseltamivir resistance-associated H275Y mutation in the neuraminidase (NA) gene of pandemic influenza A (H1N1) 2009 was occasionally reported worldwide during the 2009–2010 influenza season. A significant proportion of those were found in immunocompromised or severely ill persons. This phenomenon remains infrequent and clear recommendations for resistance testing are lacking. Objectives Present the suggested clinical selection criteria for antiviral susceptibility testing for influenza in Canada and to describe the Ontarian experience during the 2009–2010 influenza season. Study design Using a defined algorithm, we prospectively screened for OsR with pyrosequencing and phenotypic testing during the 2009–2010 influenza season. Zanamivir resistance was screened using phenotypic and sequencing technique on selected occasions. Clinical data was gathered for the resistant cases. Results A total of 804 clinical H1N1 (2009) positive samples from Ontario were screened for oseltamivir resistance between June 2009 and March 2010. We identified oseltamivir resistance in 5 (0.6%) distinct patients aged 9–62 years. All the resistant strains bore the H275Y mutation. Susceptibility to zanamivir was maintained in all of them. Three patients harboring oseltamivir resistant strain were intensive care unit patients and four were immunocompromised. All were tested for susceptibility because of a repeat positive result for influenza A PCR. Conclusion Oseltamivir resistance was not frequent during the 2009–2010 influenza season but was identified with a systematic and prospective approach to resistance testing. In order to be as sensitive as possible in the detection of those few cases, we report the suggested indications for antiviral susceptibility testing in Canada.

This study describes the validation of the TaqMan Influenza A Detection Kit v2.0 combined with an automated nucleic acid extraction method. The limit of detection of this assay was determined by probit regression (95% confidence interval) to be 2 influenza A/PR/8/34 (H1N1) virus particles per microlitre. One hundred and eleven specimens previously tested using the Seeplex RV assay and viral culture methods were tested using the TaqMan Influenza A Detection Kit. Compared to the aggregate gold-standard, the sensitivity and specificity of the TaqMan Influenza A Detection Kit were 100% (35/35) and 97% (74/76), respectively. Because of its accuracy, quick turn-around-time and lyophilized bead form, the TaqMan Influenza A Detection Kit, combined with the NucliSense easyMAG automated extraction method, constitutes a reliable protocol for influenza A diagnosis.

During the 2007-2008 influenza season global strain surveillance for antiviral resistance revealed the sudden emergence of oseltamivir resistance in influenza A H1N1 isolates. Although oseltamivir resistance rates vary from region to region, 16% of isolates tested globally were found to be oseltamivir resistant by a histidine to tyrosine mutation of residue 275 of the neuraminidase gene of influenza A. In order to implement effective resistance testing locally a novel real-time reverse-transcriptase PCR (RT-PCR) assay was developed for the detection of the H275Y mutation. To evaluate this method, 40 oseltamivir resistant and 61 oseltamivir sensitive H1N1 influenza isolates were tested using Sanger sequencing, which is the reference method for detection of resistance, pyrosequencing and the novel H275Y RT-PCR assay. In comparison to Sanger sequencing, the sensitivity and specificity of the H275Y RT-PCR assay were 100% (40/40) and 100% (61/61) respectively, while the sensitivity and specificity of pyrosequencing were 100% (40/40) and 97.5% (60/61) respectively. Although all three methods were effective in detecting the H275Y mutation associated with oseltamivir resistance, the H275Y RT-PCR assay was the most rapid and could easily be incorporated into an influenza subtyping protocol.

Background Mycoplasma pneumoniae and Chlamydophila pneumoniae are major causes of lower and upper respiratory infections that are difficult to diagnose using conventional methods such as culture. The ProPneumo-1 (Prodesse, Waukesha, WI) assay is a commercial multiplex real-time PCR assay for the simultaneous detection of M. pneumoniae and/or C. pneumoniae DNA in clinical respiratory samples. Objective The aim of this study was to evaluate the sensitivity and specificity of the ProPneumo-1, a newly developed commercial multiplex real-time PCR assay. Methods A total of 146 clinical respiratory specimens, collected from 1997 to 2007, suspected of C. pneumoniae or M. pneumoniae infections were tested retrospectively. Nucleic acid was extracted using an automated NucliSense easyMag (bioMerieux, Netherlands). We used a "Home-brew" monoplex real-time assay as the reference method for the analysis of C. pneumoniae and culture as the reference method for the analysis of M. pneumoniae. For discordant analysis specimens were re-tested using another commercial multiplex PCR, the PneumoBacter-1 assay (Seegene, Korea). Results Following discordant analysis, the sensitivity of the ProPneumo-1 assay for pathogens, C. pneumoniae or M. pneumoniae, was 100%. The specificity of the ProPneumo-1 assay, however, was 100% for C. pneumoniae and 98% for M. pneumoniae. The limits of detection were 1 genome equivalent (Geq) per reaction for pathogens, M. pneumoniae and C. pneumoniae. Due to the multipex format of the ProPneumo-1 assay, we identified 5 additional positive specimens, 2 C. pneumoniae in the M. pneumoniae-negative pool and 3 M. pneumoniae in the C. pneumoniae-negative pool. Conclusion The ProPneumo-1 assay is a rapid, sensitive and effective method for the simultaneous detection of M. pneumoniae and C. pneumoniae directly in respiratory specimens.

The Seeplex RV Detection kit was used to identify specific respiratory viruses from specimens collected during respiratory outbreaks in the Greater Toronto Area from 1 September 2007 to 1 February 2008. Two hundred-thirty-one patient samples (nasopharyngeal swabs) were collected from 63 respiratory outbreaks. The distribution of outbreaks characterized by molecular means was: 30% (n=19) no identification; 52.5% (n=33) one pathogen; 14.5% (n=9) two pathogens; and 3% (n=2) three pathogens. In contrast, culture-based protocols identified pathogens in fewer outbreaks: 63 % (n=40) no identification; 35% (n=22) 1 pathogen; and 2% (n=1) 2 pathogens (p0.05). Compared to virus isolation, molecular testing identified a greater proportion of positive specimens for rhinovirus: 22% (n=51/231) vs 5% (n=12/231) (p=0.01); and RSV A/B: 12% (n=27/231) vs 5% (n=11/231) (p0.05). Superiority of the molecular assay to detect rhinovirus and RSV outbreaks compared to culture is evident from this study.

The increase in adamantine resistance in influenza A (H3N2) and the emergence of oseltamivir resistance in influenza A (H1N1) has necessitated the use of rapid methodologies to detect influenza subtype. The purpose of this study was to evaluate the CombiMatrix influenza detection system compared to the FDA approved Luminex Respiratory virus panel (RVP) assay for influenza A subtyping. Verification of the CombiMatrix influenza detection system was carried out using the Luminex RVP assay as a reference method. A limit of detection (LOD) series was performed using the Luminex and CombiMatrix systems with both influenza A H3N2 and H1N1 viruses. Seventy-five clinical specimens were used in the study. Of these, 16 were influenza A (H3N2) positive and five were influenza A (H1N1) positive. Fifty-four specimens were influenza A negative or "no call" (inconclusive) or could not be subtyped. The LOD of the Luminex RVP assay was found to be 0.3 TCID50s/mL for influenza A (H3N2) and 16 TCID50s/mL for influenza A (H1N1). The LOD of the CombiMatrix influenza detection system was 200 TCID50s/mL for influenza A (H3N2) and 16 000 TCID50s/mL for influenza A (H1N1). The sensitivity of the CombiMatrix influenza detection system was 95.2% and the specificity was 100%. The CombiMatrix influenza detection system is an effective methodology for influenza A subtype analysis, specifically in laboratories with a constrained budget or limited molecular capabilities.

This study describes the prevalence of culture-positive adenovirus serotypes in culture-positive respiratory specimens sent to the Central Public Health Laboratory, Toronto, Ontario, Canada for the period September 2007–June 2008. Total nucleic acid was extracted from virus cultures using an automated extraction method followed by polymerase chain reaction and Sanger sequencing of the adenovirus hexon gene hypervariable region 7. 73% of specimens (n = 70) were from patients ≤ 4 years of age. Of the 96 adenovirus isolates, the most common identified serotypes were serotype 3 (n = 44, 46%), serotype 2 (n = 25, 26%), serotype 1 (n = 17, 18%), and serotype 21 (n = 5, 5%). Adenovirus serotype 14 was not found in this study group. The leading serotype, Ad3, was identified throughout the duration of the study period. Molecular methods allow for the determination of circulating adenovirus serotypes and be used to document the spread of highly virulent adenoviral serotypes into a region.