Detection of enterovirus D68 in Canadian laboratories.

The recent emergence of a severe respiratory disease caused by enterovirus D68 prompted investigation into whether Canadian hospital and provincial laboratories can detect this virus using commercial and laboratory-developed assays. This study demonstrated analytical sensitivity differences between commercial and laboratory-developed assays for the detection of enterovirus D68.

Physcomitrium patens (Hedw.) Mitt. growing in the dark defaults to an auxin- and cytokinin-independent developmental programme.

Auxin and cytokinin partially restore Physcomitrium (formerly Physcomitrella ) patens gametophores that have developed in the dark to a form more typical of those grown in light. Auxin synthesis and/or transport in gametophores decrease with time spent in the dark. Auxin synthesis resumes in the apices of dark-grown gametophores upon their return to the light. Red light and to a lesser extent blue light are sufficient for this. The mas and GH3 promoters are both auxin-inducible but respond differentially to spatial cues.

whole-genome sequence of livestock-associated st398 methicillin-resistant staphylococcus aureus Isolated from Humans in Canada.

Despite reports of high colonization rates of ST398 livestock-associated methicillin-resistant Staphylococcus aureus (LA-MRSA) among pigs and pig farmers, the incidence of LA-MRSA infection in the general population in Canada appears to be rare in comparison to that in some European countries. In this study, the complete genome sequence of a Canadian representative LA-MRSA isolate (08BA02176) from a human postoperative surgical site infection was acquired and compared to the sequenced genome of an LA-MRSA isolate (S0385) from Europe to identify genetic traits that may explain differences in the success of these particular strains in some locales.

High rates of Staphylococcus aureus USA400 infection, Northern Canada.

Surveillance of Staphylococcus aureus infections in 3 northern remote communities of Saskatchewan was undertaken. Rates of methicillin-resistant infections were extremely high (146-482/10,000 population), and most (98.2%) were caused by USA400 strains. Although USA400 prevalence has diminished in the United States, this strain is continuing to predominate throughout many northern communities in Canada.

Livestock-associated methicillin-resistant Staphylococcus aureus sequence type 398 in humans, Canada.

Rates of colonization with livestock-associated methicillin-resistant Staphylococcus aureus (MRSA) sequence type 398 have been high for pigs and pig farmers in Canada, but prevalence rates for the general human population are unknown. In this study, 5 LA-MRSA isolates, 4 of which were obtained from skin and soft tissue infections, were identified from 3,687 tested MRSA isolates from persons in Manitoba and Saskatchewan, Canada. Further molecular characterization determined that these isolates all contained staphylococcal cassette chromosome (SCC) mecV, were negative for Panton-Valentine leukocidin, and were closely related by macrorestriction analysis with the restriction enzyme Cfr91. The complete DNA sequence of the SCCmec region from the isolate showed a novel subtype of SCCmecV harboring clustered regularly interspaced short palindromic repeats and associated genes. Although prevalence of livestock-associated MRSA seems to be low for the general population in Canada, recent emergence of infections resulting from this strain is of public health concern.

Rapid detection of Norovirus based on an automated extraction protocol and a real-time multiplexed single-step RT-PCR.

BACKGROUND: Molecular diagnosis of Norovirus infection can be a complex multistep process, which requires significant user intervention and expertise, and is not amenable to automation without extensive validation and optimization. OBJECTIVES: To develop a real-time multiplexed RT-PCR assay with automated sample preparation that requires only a single-step and a single-tube for reverse transcription, amplification, and detection while exceeding the sensitivity of conventional PCR for broad-spectrum Norovirus detection. STUDY DESIGN: Limit of detection was assessed against dilutions of clinical specimens. Fifty archived extractions were used to compare TaqMan sensitivity with either a separate RT using random primers or a single-step RT-PCR. The sensitivity of the novel assay was compared with conventional RT-PCR using 100 specimens from gastroenteritis cases. RESULTS: Automated extraction reduced RNA recovery by 0.5 logs compared to manual extraction but was more effective at removing PCR inhibitors from stool specimens. The optimized single-step real-time RT-PCR demonstrated no reduction in sensitivity. Together, the sensitivity of the novel assay was 19% higher than manual extraction with conventional RT-PCR. CONCLUSIONS: A semi-automated and simplified molecular diagnostic protocol for the rapid detection of Norovirus has been achieved. PCR inhibitors are present in human fecal specimens and cause a significant problem for Norovirus detection by RT-PCR.

Draft Genome Sequence of Streptococcus pyogenes Strain 06BA18369, a Human Pathogen Associated with Skin and Soft Tissue Infections in Northern Canada.

We report the draft sequence of Streptococcus pyogenes 06BA18369 (emm type 41.2, sequence type 579 [ST579]), isolated from a skin and soft tissue infection (SSTI) mixed with Staphylococcus aureus. This genome provides insight into the genetic composition of S. pyogenes strains associated with mixed SSTIs.

Draft Genome Sequence of Methicillin-Susceptible Staphylococcus aureus Strain 06BA18369, a Pathogen Associated with Skin and Soft Tissue Infections in Northern Saskatchewan, Canada.

Here, we announce the draft sequence of a representative methicillin-susceptible Staphylococcus aureus (MSSA) isolate (06BA18369) whose strain type (spa type t311) was commonly isolated from skin and soft tissue coinfections with Streptococcus pyogenes. This strain sequence provides insight into a highly successful community-associated MSSA strain type.

Characterization of Escherichia coli urinary tract infection isolates in remote northern Saskatchewan communities: the Northern Antibiotic Resistance Partnership.

Antimicrobial resistance is a growing concern especially in many remote northern communities of Canada where antimicrobials are liberally used. In this study, 1418 Escherichia coli urinary tract infection (UTI) isolates, obtained over a 2.5-year period (October 2005-March 2008), from 3 remote northern sites in Saskatchewan, Canada, were identified. Antimicrobial susceptibility testing of the first 544 clinically significant isolates revealed high prevalence of resistance to trimethoprim-sulfamethoxazole (TMP-SXT) (30.7%). Pulsed-field gel electrophoresis (PFGE) of 165 TMP-SXT-resistant isolates revealed a heterogeneous population. Multilocus sequence typing identified 7 STs from 9 identified PFGE clusters, which included separate PFGE clusters of fluoroquinolone-resistant and -susceptible ST131 isolates. The majority of TMP-SXT-resistant isolates (85.5%) were found to carry class 1 integrons, and plasmids from 62 (81%) of 77 representative isolates were successfully transformed into E. coli DH10B. Overall, ampicillin was the most common plasmid-encoded resistance phenotype transferred with TMP-SXT at 60% (37/62). Further characterization of 52 plasmids by restriction fragment length polymorphism and replicon typing revealed the presence of many plasmid lineages, suggesting that the elevated rates of TMP-SXT resistance in these communities are most likely attributed to the horizontal transfer of class 1 integrons. Results from this study emphasize the importance of continued surveillance of remote northern communities in order to optimize the efficacy of empiric UTI treatment.

Comparison of Shiga toxin-producing Escherichia coli detection methods using clinical stool samples.

Molecular diagnostic tools capable of identifying Shiga toxin-specific genetic determinants in stool specimens permit an unbiased approach to detect Shiga toxin-producing Escherichia coli (STEC) in clinical samples and can indicate when culture-based isolation methods are required. It is increasingly recognized that clinically relevant STEC are not limited to the singular O157 serotypes, and therefore diagnostic assays targeting toxin-encoding determinants must be able to account for any genetic variation that exists between serotypes. In this study conventional PCR and four real-time PCR assays (HybProbe, TaqMan, SYBR Green, and LUX) targeting the stx1 and stx2 Shiga toxin coding sequences were used to identify STEC in enriched stool samples (n = 36) and a panel of O157 and non-O157 strains (n = 64). PCR assays targeting stx1 and stx2 had variable specificity and sensitivity values with enriched stool samples. Molecular assays using DNA from pure cultures revealed that some primers were not sensitive to all stx2 variants. This evaluation concluded that the TaqMan-based probes were most appropriate in high throughput clinical diagnostic laboratories in consideration of cost, turn around time, and assay performance.

High-throughput qualitative multiplex 5' nuclease assay using post-only PCR analysis.

Homogenous fluorescence PCR assays offer distinct advantages for qualitative testing and are gaining immense popularity in fields like diagnostic microbiology. To meet the demand of high-volume laboratories, we developed a protocol for qualitative multiplex 5' nuclease assays using post-only PCR analysis. This novel approach overcomes throughput problems encountered with the established methods for TaqMan detection on the ABI PRISM 7700 Sequence Detection system and permits off-site TaqMan PCR, which can be analyzed several days after the reactions are completed. We have validated this novel protocol using an assay for the identification of Bordetella pertussis against real-time and plate-read analysis methods and have shown that our proposed protocol produces no difference in qualitative calls.

Development of a triplex real-time PCR assay for detection of Panton-Valentine leukocidin toxin genes in clinical isolates of methicillin-resistant Staphylococcus aureus.

Community-associated methicillin-resistant Staphylococcus aureus harboring Panton-Valentine leukocidin (PVL) genes is an emerging pathogen. A novel real-time PCR assay for identification of MRSA isolates containing PVL was developed. The PVL assay was used in a triplex format allowing simultaneous amplification of mecA, nuc, and PVL genes in 614 clinical isolates. This assay facilitates the rapid identification of PVL-positive isolates of MRSA.

Rapid genotyping of hepatitis C virus by primer-specific extension analysis.

Quick and accurate genotyping of hepatitis C virus (HCV) is becoming increasingly important for clinical management of chronic infection and as an epidemiological marker. Furthermore, the incidence of HCV infection with mixed genotypes has clinical significance that is not addressed by most genotyping methods. We have developed a fluorescence-based genotyping assay called primer-specific extension analysis (PSEA) for the most prevalent HCV genotypes and have demonstrated the capacity of PSEA-HCV for detecting mixed-genotype HCV infections. PSEA-HCV detects genotype-specific sequence differences in the 5' untranslated region of HCV in products amplified by the COBAS AMPLICOR HCV Test, v2.0. Simulated mixed HCV infection of plasma with RNase-resistant RNA controls demonstrates that PSEA-HCV can detect as many as five genotypes in one specimen. Furthermore, in dual-genotype simulations, PSEA-HCV can unequivocally detect both genotypes, with one genotype representing only 3.1% of the mixture (313/10,000 IU in starting plasma). Compared to INNO-LiPA HCV II, both assays determined the same genotype for 191/199 (96%) patient specimens (175 subtype and 16 genotype-only identifications). Following the initial evaluation, PSEA-HCV was used routinely to genotype HCV from patient specimens submitted to our laboratory (n=312). Seventeen (5.4%) mixed infections were identified. The distribution of single-infection HCV genotypes in our population was 60.9% type 1 (n=190), 12.8% type 2 (n=40), 20.2% type 3 (n=63), 0.3% type 4 (n=1), and 0.3% other (n=1). In conclusion, PSEA-HCV provides an inexpensive, high-throughput screening tool for rapid genotyping of HCV while reliably identifying mixed HCV infections.