Steven J. Drews

Novel duplex real-time PCR assay detects Bordetella holmesii in specimens from patients with Pertussis-like symptoms in Ontario, Canada.

ABSTRACT Bordetella holmesii is a human pathogen found mainly in immunocompromised patients. A specific real-time PCR assay was developed and successfully used to identify specimens from which B. holmesii was misidentified as Bordetella pertussis and to establish the prevalence of B. holmesii in Ontario patients with pertussis-like symptoms.

Use of Bordetella pertussis BP3385 To Establish a Cutoff Value for an IS481-Targeted Real-Time PCR Assay

ABSTRACT This study utilized the Bordetella pertussis single-copy PCR target BP3385 as a means of confirming IS481 PCR-positive reactions with cycle threshold (CT) values of >35. IS481 PCRs with CT values of >35 cycles may represent PCR conditions where there is <1 CFU of B. pertussis per PCR.

Development of a novel real-time reverse-transcriptase PCR method for the detection of H275Y positive influenza A H1N1 isolates.

n Abstractn n 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.n n

Why "winter" vomiting disease? Seasonality, hydrology, and Norovirus epidemiology in Toronto, Canada.

Norovirus is a common cause of gastroenteritis, and is thought to be the causative agent in 68–90% of all gastroenteritis outbreaks. The seasonality of disease occurrence is sufficiently stereotyped to result in this disease being dubbed “winter vomiting disease.” The genesis of this seasonality has been obscure. We sought to identify environmental factors associated with Norovirus outbreaks in Toronto, Canada. We evaluated 253 outbreaks of gastroenteritis linked to Norovirus between November 2005 and March 2008. Poisson regression models were constructed to evaluate associations between average environmental exposures and case counts. A case-crossover approach was used to evaluate associations between acute changes in environment and outbreak risk. Case-crossover analysis indicated an association between low Lake Ontario temperature (≤4°C) (hazard ratio [HR], 5.61 [95% CI, 2.81–11.12]) and high flow (>2.5xa0m3/s) in the Don River (HR, 3.17 [95% CI, 2.30–4.36]), 1–7xa0days prior to case occurrence. For both exposure variables, the highest hazard ratios were found 24–48xa0h prior to case onset. Regression models provided further support for these patterns. The association between local watershed conditions and Norovirus outbreak risk suggest a source-water reservoir for this pathogen. We hypothesize that the reservoir may be maintained through the discharge of wastewater containing virus particles; wintertime seasonality may be explained by enhanced viral persistence at low temperatures.

Comparison of a commercial qualitative real-time RT-PCR kit with direct immunofluorescence assay (DFA) and cell culture for detection of influenza A and B in children

BACKGROUNDnInstitutional pandemic planning prompted a study of the molecular detection of influenza virus from respiratory specimens in children, compared to conventional diagnostics.nnnOBJECTIVEnTo evaluate the performance of a commercial qualitative real-time RT-PCR kit (rRT-PCR), the artus Influenza LC RT-PCR (Qiagen). STUDY DESIGN (METHODS): Specimens were pre-selected to include a high percentage of positives by direct immunofluorescence assay (DFA) or culture. The sensitivity and specificity of the kit for detection of influenza A and B in children were determined against the gold standard, DFA and culture. Specimens yielding discordant results between artus and the gold standard were tested against a reference rRT-PCR assay (Centers for Disease Control) to create an expanded gold standard.nnnRESULTSnWhen compared to DFA or cell culture, the sensitivity of the rRT-PCR artus kit was 96.2% and the specificity was 94%. It detected influenza RNA in 6.0% of clinical samples negative by DFA or culture. Using the expanded gold standard, the revised sensitivity was 98.7% (98.6% for influenza A and 97.6% for influenza B) and the specificity was 100%.nnnCONCLUSIONnThe artus Influenza LC RT-PCR kit is an effective alternative to virus isolation and DFA for the detection of influenza A and B in pediatric clinical specimens.

Comparison of Two Versions of the IDI-MRSA Assay Using Charcoal Swabs for Prospective Nasal and Nonnasal Surveillance Samples

ABSTRACT An updated IDI-MRSA assay version was released to address the assays low positive predictive value (PPV). A prospective analysis of two assay versions indicated no significant improvement in the PPV. Colonization by methicillin-resistant Staphylococcus aureus in 24% of patients would not have been detected if only nasal samples had been tested, as approved, by this molecular method.

Molecular characterization of drug-resistant Mycobacterium tuberculosis isolates from Ontario, Canada

OBJECTIVESnOntario bears the greatest burden of tuberculosis in Canada, with 40% of all cases and 60% of multidrug-resistant cases. The purpose of this study was to genotypically characterize isoniazid- and rifampicin-resistant isolates and compare these results with phenotypic drug susceptibility testing data. This is the first Canadian study to examine gene mutations that contribute to multidrug-resistant tuberculosis.nnnMETHODSnA total of 751 tuberculosis isolates were tested for drug resistance using phenotypic antimicrobial susceptibility testing methods. Isolates were then characterized using molecular methods. Following DNA extraction, PCR amplification and sequence analysis were performed on the rifampicin resistance region of rpoB, as well as the region surrounding katG315 and the inhA promoter region associated with isoniazid resistance.nnnRESULTSnEighteen different mutation types were found in the rpoB region of rifampicin-resistant isolates. Isolates with mutations at residues rpoB531 (64.1%), rpoB526 (15.2%) and rpoB516 (8.7%) were the most common. In addition, an insertion was found at residue 514. Three phenotypically rifampicin-resistant isolates (3.3%) were genotypically wild-type. In isoniazid-resistant strains, mutations were found most commonly at katG315 (45.4%) as well as at the inhA promoter region (28.6%). Thirty-nine isolates (25.3%) were phenotypically isoniazid-resistant but genotypically wild-type. The katG315 mutation was statistically associated with multidrug-resistant isolates.nnnCONCLUSIONSnThis study expands the knowledge of mutations that potentially contribute to drug resistance in tuberculosis and lays the foundation for developing molecular-based tests to determine drug resistance in clinical tuberculosis isolates.

Differential patterns of amantadine-resistance in influenza A (H3N2) and (H1N1) isolates in Toronto, Canada.

BACKGROUNDnMolecular methods were used to characterize influenza A (H1N1) and (H3N2) strains and to identify amantadine-resistance.nnnOBJECTIVESnTo compare proportions of amantadine-resistant influenza A (H1N1) and (H3N2) isolates in the Greater Toronto Area.nnnSTUDY DESIGNnIsolates of influenza A (H1N1) and (H3N2) were strain typed using molecular methods. Pyrosequencing for point mutations in the transmembrane domain of the M2 proton channel was undertaken. Proportions of amantadine-resistant and susceptible isolates were compared using the The Fishers exact test.nnnRESULTSn96% of the 49 influenza A (H3N2) isolates and none of the influenza A (H1N1) tested carried a point mutation in the M gene coding for the M2 protein. Influenza A (H3N2) isolates were more likely to carry an amantadine-resistance associated mutation than influenza A (H1N1) isolates (Fisherss exact test, P<0.0001).nnnCONCLUSIONSn: Characterization of amantadine-resistance in influenza A (H1N1) isolates should utilize a variety of different methods including sub-typing, strain typing, and direct sequencing for point mutations associated with amantadine-resistance.

Characterization of culture-positive adenovirus serotypes from respiratory specimens in Toronto, Ontario, Canada: September 2007–June 2008

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.

Genetic microheterogeneity of emerging H275Y influenza virus A (H1N1) in Toronto, Ontario, Canada from the 2007–2008 respiratory season

BACKGROUNDnThe H275Y mutation (H274Y in N2 numbering) in the neuraminidase (NA) gene (segment 6) of the influenza virus A (H1N1) genome is linked to oseltamivir resistance.nnnOBJECTIVESnTo determine the percentage of influenza virus A (H1N1) isolates that carry the H275Y mutation in the NA gene in Toronto, Ontario, Canada and to characterize select oseltamivir resistant and susceptible isolates using sequence analysis.nnnSTUDY DESIGNnSanger sequencing was used to determine strain type and H275Y mutations based on partial sequencing of the hemagglutinin (HA) (segment 4) and NA genes. Mutations in the NS1 gene (segment 8) were determined by Sanger sequencing and pyrosequencing. Statistical analysis of demographics and proportions of H275 and H275Y isolates with mutations was carried out using chi(2) analyses.nnnRESULTSnThe HA gene of influenza virus A (H1N1) isolates collected during the 2007-2008 respiratory season was most like influenza A/Brisbane/59/2007, Clade 2, subclade B. Seventeen percent of these isolates possessed the H275Y NA mutation associated with oseltamivir resistance. H275Y isolates were more likely than H275 isolates to have the mutations A209T and R224G in NS1 (chi(2)=284.9, df=2, p<0.0001).nnnCONCLUSIONSnDuring the 2007-2008 influenza season in Toronto, Ontario, Canada, 17% of influenza virus A (H1N1) isolates carried the H275Y mutation associated with oseltamivir resistance. These H275Y isolates were more likely than H275 isolates to exhibit unique microheterogeneity in the gene encoding the NS1 protein.