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1.  Utility of Specimens Positive for Neisseria gonorrhoeae by the Aptima Combo 2 Assay for Assessment of Strain Diversity and Antibiotic Resistance 
Journal of Clinical Microbiology  2013;51(12):4156-4160.
In our jurisdiction, the Aptima Combo 2 assay (Gen-Probe, Inc.) is used to detect Neisseria gonorrhoeae from specimens collected at clinics for sexually transmitted infections (STI) and from select community patients. In addition, swabs are also collected for N. gonorrhoeae culture, susceptibility testing, and sequence typing (ST). Since only a small proportion of samples from provincial cases undergo culture, the available trends in antimicrobial susceptibility and predominant strain types may not be representative of all N. gonorrhoeae infections. Due to the limitations facing the use of N. gonorrhoeae culture to understand these trends in the general community, we performed a molecular analysis for markers of cephalosporin resistance and ST determination by using nucleic acid extracts of specimens sent for Aptima testing. Thirty-four samples submitted for both Aptima testing and N. gonorrhoeae culture from the same anatomic location (within 24 h) were included in the study. Sequence type was determined based on the sequence of the por and tbpB genes, and amino acid changes in the PBP 2 protein, encoded by the penA gene, were considered representative for the assessment of antimicrobial susceptibility. Sequence identity of 100% was observed between the sequences obtained from Aptima-analyzed samples and culture samples. Sequencing results showed an association between decreased susceptibility to extended-spectrum cephalosporins (ESCds), tbp allele 110, ST 1407, and amino acid changes (G545S, I312M, and V316T) in the PBP 2 protein. Our data, generated based on a few representative genes, suggest that gonococcal samples positive by Aptima testing can be used to determine single nucleotide polymorphisms associated with ESCds and the sequence type based on molecular strain typing. Confirmation of these findings may obviate the need for gonorrhea culture in the future.
doi:10.1128/JCM.01694-13
PMCID: PMC3838053  PMID: 24108603
2.  Detection of Influenza H7N9 Virus: All Molecular Tests Are Not Equal 
Journal of Clinical Microbiology  2013;51(11):3835-3838.
The recent emergence of influenza A virus (H7N9) emphasizes the need for its rapid detection. While commercial nucleic acid amplification tests (NAATs) are commonly used to detect seasonal influenza virus, this study demonstrated that the analytical sensitivity of commercial assays is highly variable compared to that of CDC-based in-house NAATs for the detection of H7N9.
doi:10.1128/JCM.01808-13
PMCID: PMC3889775  PMID: 23966485
3.  Full-Genome Analysis of Avian Influenza A(H5N1) Virus from a Human, North America, 2013 
Emerging Infectious Diseases  2014;20(5):887-891.
Full-genome analysis was conducted on the first isolate of a highly pathogenic avian influenza A(H5N1) virus from a human in North America. The virus has a hemagglutinin gene of clade 2.3.2.1c and is a reassortant with an H9N2 subtype lineage polymerase basic 2 gene. No mutations conferring resistance to adamantanes or neuraminidase inhibitors were found.
doi:10.3201/eid2005.140164
PMCID: PMC4012823  PMID: 24755439
avian influenza; H5N1 subtype; highly pathogenic avian influenza; HPAI; North America; reassortant; influenza A; full genome; human; H9N2 subtype; viruses; influenza; genome analysis; Canada
4.  Bordetella pertussis in sporadic and outbreak settings in Alberta, Canada, July 2004 – December 2012 
Background
ProvLab Alberta provides all laboratory testing for Bordetella pertussis including sporadic cases and outbreak investigations through collaborations with provincial public health partners. We describe B. pertussis activity in Alberta from July 2004 to December 2012.
Methods
Laboratory testing for pertussis was analyzed using interpreted laboratory data that was generated by DIAL, a secure web-based platform. Duplicate specimens from the same individual ≤90 days were excluded to generate a case-based dataset. Immunization status of confirmed pertussis cases from the provincial immunization repository was reviewed.
Results
Overall, 7.1% of suspected pertussis cases tested positive with a higher positivity rate in outbreak as compared to sporadic setting. Annual variations in sporadic pertussis cases were observed across the province with higher positivity rates in 2005, 2008, 2009 and 2012. A significantly higher positivity rate was observed in a northern region of Alberta. While the positivity rate in sporadic setting was highest in adolescents aged 10 to <15 years old (14.8%), population-based disease burden was highest in young children <5 years old. Of the 81.6% (n = 1,348) pertussis cases with immunization records, 48.3% were up-to-date with immunization. The pertussis cases that were up-to-date with their immunization were older (median age 12.9 years) as compared to those with incomplete (median age 9.7 years) or no pertussis immunization (median age 3.8 years).
Conclusions
Cyclic pattern of annual pertussis activity with geographic variation was observed in Alberta with no obvious case finding effect from outbreak investigations. The high positivity rates in adolescents suggested an underestimation of disease burden in this age group.
doi:10.1186/1471-2334-14-48
PMCID: PMC3931923  PMID: 24476570
Pertussis; Outbreaks; Epidemiology
5.  Use of an Innovative Web-Based Laboratory Surveillance Platform to Analyze Mixed Infections Between Human Metapneumovirus (hMPV) and Other Respiratory Viruses Circulating in Alberta (AB), Canada (2009–2012) 
Viruses  2012;4(11):2754-2765.
We investigated the proportions of mono vs. mixed infections for human metapneumovirus (hMPV) as compared to adenovirus (ADV), four types of coronavirus (CRV), parainfluenza virus (PIV), RSV, and enterovirus/rhinovirus (ERV) in Alberta, Canada. Using the Data Integration for Alberta Laboratories (DIAL) platform, 26,226 respiratory specimens at ProvLab between 1 July 2009 and 30 June 2012 were selected and included in the study. Using the Respiratory Virus Panel these specimens tested positive for one or more respiratory virus and negative for influenza A and B. From our subset hMPV was the fourth most common virus (n=2,561) with 373 (15%) identified as mixed infection using DIAL. Mixed infection with hMPV was most commonly found in infants less than 6 months old and ERV was most commonly found in mixed infection with hMPV (230/373, 56%) across all age groups. The proportion of mixed-infection vs. mono-infection was highest for ADV (46%), followed by CRV 229E (32%), CRV HKU1 (31%), CRV NL63 (28%), CRV OC43 (23%), PIV (20%), RSV (17%), hMPV (15%) and ERV (13%). hMPV was significantly more likely to be identified in mono infection as compared with ADV, CRV, PIV, and RSV with the exception of ERV [p<0.05].
doi:10.3390/v4112754
PMCID: PMC3509671  PMID: 23202503
hMPV; co-infection; testing; epidemiology; respiratory
6.  Comparison of the Luminex xTAG Respiratory Viral Panel with xTAG Respiratory Viral Panel Fast for Diagnosis of Respiratory Virus Infections▿ 
Journal of Clinical Microbiology  2011;49(5):1738-1744.
Nucleic acid tests are sensitive and specific and provide a rapid diagnosis, making them invaluable for patient and outbreak management. Multiplex PCR assays have additional advantages in providing an economical and comprehensive panel for many common respiratory viruses. Previous reports have shown the utility of the xTAG respiratory viral panel (RVP) assay manufactured by Luminex Molecular Diagnostics for this purpose. A newer generation of this kit, released in Canada in early 2010, is designed to simplify the procedure and reduce the turnaround time by about 24 h. The assay methodology and targets included in this version of the kit are different; consequently, the objective of this study was to compare the detection of a panel of respiratory viral targets using the older Luminex xTAG RVP (RVP Classic) assay with that using the newer xTAG RVP Fast assay. This study included 334 respiratory specimens that had been characterized for a variety of respiratory viral targets; all samples were tested by both versions of the RVP assay in parallel. Overall, the RVP Classic assay was more sensitive than the RVP Fast assay (88.6% and 77.5% sensitivities, respectively) for all the viral targets combined. Targets not detected by the RVP Fast assay included primarily influenza B virus, parainfluenza virus type 2, and human coronavirus 229E. A small number of samples positive for influenza A virus, respiratory syncytial virus B, human metapneumovirus, and parainfluenza virus type 1 were not detected by the RVP Classic assay and in general had low viral loads.
doi:10.1128/JCM.02090-10
PMCID: PMC3122679  PMID: 21411570
7.  Pertussis resurgence in Toronto, Canada: a population-based study including test-incidence feedback modeling 
BMC Public Health  2011;11:694.
Background
Pertussis continues to challenge medical professionals; recently described increases in incidence may be due to age-cohort effects, vaccine effectiveness, or changes in testing patterns. Toronto, Canada has recently experienced increases in pertussis incidence, and provides an ideal jurisdiction for evaluating pertussis epidemiology due to centralized testing. We evaluated pertussis trends in Toronto using all available specimen data, which allowed us to control for changing testing patterns and practices.
Methods
Data included all pertussis culture and PCR test records for Greater Toronto from 1993 to 2007. We estimated incidence trends using Poisson regression models; complex relationships between disease incidence and test submission were explored with vector autoregressive models.
Results
From 1993 to 2007, 26988 specimens were submitted for testing; 2545 (9.4%) were positive. Pertussis incidence was 2 per 100,000 from 1993 to 2004 and increased to 10 per 100,000 from 2005-2007, with a concomitant 6-fold surge in test specimen submissions after the introduction of a new, more sensitive PCR assay. The relative change in incidence was less marked after adjustment for testing volumes. Bidirectional feedbacks between test positivity and test submissions were identified.
Conclusions
Toronto's recent surge in pertussis reflects a true increase in local disease activity; the apparent size of the outbreak has likely been magnified by increasing use of pertussis testing by clinicians, and by improved test sensitivity since 2005. These findings may be applicable to changes in pertussis epidemiology that have been noted elsewhere in North America.
doi:10.1186/1471-2458-11-694
PMCID: PMC3189138  PMID: 21899765
8.  When should a diagnosis of influenza be considered in adults requiring intensive care unit admission? Results of population-based active surveillance in Toronto 
Critical Care  2011;15(4):R182.
Introduction
There is a paucity of data about the clinical characteristics that help identify patients at high risk of influenza infection upon ICU admission. We aimed to identify predictors of influenza infection in patients admitted to ICUs during the 2007/2008 and 2008/2009 influenza seasons and the second wave of the 2009 H1N1 influenza pandemic as well as to identify populations with increased likelihood of seasonal and pandemic 2009 influenza (pH1N1) infection.
Methods
Six Toronto acute care hospitals participated in active surveillance for laboratory-confirmed influenza requiring ICU admission during periods of influenza activity from 2007 to 2009. Nasopharyngeal swabs were obtained from patients who presented to our hospitals with acute respiratory or cardiac illness or febrile illness without a clear nonrespiratory aetiology. Predictors of influenza were assessed by multivariable logistic regression analysis and the likelihood of influenza in different populations was calculated.
Results
In 5,482 patients, 126 (2.3%) were found to have influenza. Admission temperature ≥38°C (odds ratio (OR) 4.7 for pH1N1, 2.3 for seasonal influenza) and admission diagnosis of pneumonia or respiratory infection (OR 7.3 for pH1N1, 4.2 for seasonal influenza) were independent predictors for influenza. During the peak weeks of influenza seasons, 17% of afebrile patients and 27% of febrile patients with pneumonia or respiratory infection had influenza. During the second wave of the 2009 pandemic, 26% of afebrile patients and 70% of febrile patients with pneumonia or respiratory infection had influenza.
Conclusions
The findings of our study may assist clinicians in decision making regarding optimal management of adult patients admitted to ICUs during future influenza seasons. Influenza testing, empiric antiviral therapy and empiric infection control precautions should be considered in those patients who are admitted during influenza season with a diagnosis of pneumonia or respiratory infection and are either febrile or admitted during weeks of peak influenza activity.
doi:10.1186/cc10331
PMCID: PMC3387625  PMID: 21798012
9.  Self-Collected Mid-Turbinate Swabs for the Detection of Respiratory Viruses in Adults with Acute Respiratory Illnesses 
PLoS ONE  2011;6(6):e21335.
Background
The gold standard for respiratory virus testing is a nasopharyngeal (NP) swab, which is collected by a healthcare worker. Midturbinate (MT) swabs are an alternative due to their ease of collection and possible self-collection by patients. The objective of this study was to compare the respiratory virus isolation of flocked MT swabs compared to flocked NP swabs.
Methods
Beginning in October 2008, healthy adults aged 18 to 69 years were recruited into a cohort and followed up for symptoms of influenza. They were asked to have NP and MT swabs taken as soon as possible after the onset of a fever or two or more respiratory symptoms with an acute onset. The swabs were tested for viral respiratory infections using Seeplex® RV12 multiplex PCR detection kit. Seventy six pairs of simultaneous NP and MT swabs were collected from 38 symptomatic subjects. Twenty nine (38%) of these pairs were positive by either NP or MT swabs or both. Sixty nine (91%) of the pair results were concordant. Two samples (3%) for hCV OC43/HKU1 and 1 sample (1%) for rhinovirus A/B were positive by NP but negative by MT. One sample each for hCV 229E/NL63, hCV OC43/HKU1, respiratory syncytial virus A, and influenza B were positive by MT but negative by NP.
Conclusions
Flocked MT swabs are sensitive for the diagnosis of multiple respiratory viruses. Given the ease of MT collection and similar results between the two swabs, it is likely that MT swabs should be the preferred method of respiratory cell collection for outpatient studies. In light of this data, larger studies should be performed to ensure that this still holds true and data should also be collected on the patient preference of collection methods.
doi:10.1371/journal.pone.0021335
PMCID: PMC3121745  PMID: 21731708
10.  Determination of the relative economic impact of different molecular-based laboratory algorithms for respiratory viral pathogen detection, including Pandemic (H1N1), using a secure web based platform 
Virology Journal  2011;8:277.
Background
During period of crisis, laboratory planners may be faced with a need to make operational and clinical decisions in the face of limited information. To avoid this dilemma, our laboratory utilizes a secure web based platform, Data Integration for Alberta Laboratories (DIAL) to make near real-time decisions.
This manuscript utilizes the data collected by DIAL as well as laboratory test cost modeling to identify the relative economic impact of four proposed scenarios of testing for Pandemic H1N1 (2009) and other respiratory viral pathogens.
Methods
Historical data was collected from the two waves of the pandemic using DIAL. Four proposed molecular testing scenarios were generated: A) Luminex respiratory virus panel (RVP) first with/without US centers for Disease Control Influenza A Matrix gene assay (CDC-M), B) CDC-M first with/without RVP, C) RVP only, and D) CDC-M only. Relative cost estimates of different testing algorithm were generated from a review of historical costs in the lab and were based on 2009 Canadian dollars.
Results
Scenarios A and B had similar costs when the rate of influenza A was low (< 10%) with higher relative cost in Scenario A with increasing incidence. Scenario A provided more information about mixed respiratory virus infection as compared with Scenario B.
Conclusions
No one approach is applicable to all conditions. Testing costs will vary depending on the test volume, prevalence of influenza A strains, as well as other circulating viruses and a more costly algorithm involving a combination of different tests may be chosen to ensure that tests results are returned to the clinician in a quicker manner. Costing should not be the only consideration for determination of laboratory algorithms.
doi:10.1186/1743-422X-8-277
PMCID: PMC3123288  PMID: 21645365
influenza; testing; relative comparisons; test algorithms; economic impact
11.  The Ontario Universal Typing of Tuberculosis (OUT-TB) Surveillance Program – what it means to you 
BACKGROUND:
Tuberculosis (TB) is a serious disease that is transmitted primarily by the airborne route. Effective disease control and outbreak management requires the timely diagnosis, isolation and treatment of infected individuals with active disease; contact tracing to identify secondary cases likely to benefit from treatment of latent infection; and laboratory identification or confirmation of epidemiologically linked cases. TB genotyping enables the comparison of Mycobacterium tuberculosis complex (MTBC) strains and the identification of cases that may or may not be linked. The increased availability of molecular methods for genotyping has allowed for greater discrimination of MTBC strains and greatly enhanced understanding of TB transmission patterns.
OBJECTIVE:
To improve TB surveillance and control in Ontario, the Public Health Laboratories of the Ontario Agency for Health Protection and Promotion has introduced the Ontario Universal Typing of Tuberculosis (OUT-TB) Surveillance Program.
METHODS:
The first isolate from every new TB case will be genotyped with two rapid molecular methods: spoligotyping and mycobacterial interspersed repetitive unit-variable-number tandem repeat typing. MTBC isolates with nonunique genotypes and, thus, potentially linked to other TB cases, will also be genotyped by IS6110 restriction fragment length polymorphism analysis.
CONCLUSION:
By providing TB control programs using these new genotyping tools, and using traditional and new case investigation methods (eg, social network analysis), this new program will provide a clearer picture of TB in Ontario, and permit more effective use of public health resources and improve disease control.
PMCID: PMC2900146  PMID: 20617215
Genotyping; Public health; Tuberculosis
12.  Laboratory based surveillance of travel-related Shigella sonnei and Shigella flexneri in Alberta from 2002 to 2007 
Between 2002 and 2007, travel related cases of Shigella sonnei and S. flexneri in Alberta, Canada were acquired from Central America, the Indian subcontinent and North America. Of this group, resistance to ciprofloxacin and nalidixic acid was identified in isolates from patients who had travelled to the Indian subcontinent. This study provides a Canadian perspective to a growing body of literature linking ciprofloxacin and nalidixic acid resistance to travel to the Indian subcontinent.
Shigella is a common cause of diarrheal illness in North America with a rate of 2.0 per 100,000 in Canada [1] and a rate of 3.2 per 100,000 in the United States [2,3]. Imported cases of Shigella infections have been reported in developed countries following travel to a foreign or developing country [4,5] and may be impacted by factors including socio-economic factors [6], food distribution networks [5] and microbiologic factors [7]. Across multiple geographic regions, high rates of antimicrobial resistance to multiple agents (e.g. sulfonamides, tetracycline, chloramphenicol, ampicillin, and trimethoprim-sulfamethoxazole) have limited the choices for empiric antimicrobial therapy required to manage Shigella infections and reduce fecal excretion of the bacteria [8-10] with descriptions of shifting species dominance and changes in antimicrobial susceptibility [10,11]. Generally, Shigella flexneri and Shigella sonnei are the dominant species and are heavily impacted by changes in antimicrobial susceptibility [12,13].
This study identifies the global regions associated with travel-related cases of S. flexneri and S. sonnei in Alberta, Canada and compares antibiotic resistance patterns of these isolates for 2002 to 2007 inclusive.
Specimens collected 2002-2007 (inclusive) from S. flexneri and S. sonnei infections in Alberta, Canada were included for study. Data collected at time of specimen submission included: date of specimen collection, outbreak association if present, travel history and antibiogram (data source-ProvLab Information Systems; Communicable Disease Report at Alberta Health and Wellness). Outbreaks were defined by public health officials as ≥ 2 epidemiologically related cases. Each outbreak was assigned a unique incident number. Repeat isolates received within six months of original case infections were excluded. Only one representative case for each outbreak was included, unless the isolates had different antibiotic susceptibility patterns. Based on travel history the origin of an isolate was grouped into corresponding regions and continents. Regions included in the study represented major travel destinations for individuals living in Canada. Domestic exposures were defined as "travel within North America."
doi:10.1186/1744-8603-6-20
PMCID: PMC2988768  PMID: 21040566
13.  Epidemiology of Invasive Meningococcal Disease with Decreased Susceptibility to Penicillin in Ontario, Canada, 2000 to 2006▿  
Neisseria meningitidis has been relatively slow to acquire resistance to penicillin. We previously reported an increase in the incidence of invasive meningococcal disease (IMD) strains with decreased susceptibility to penicillin (DSP) in Ontario. Our objectives were to evaluate trends in IMD with DSP, to identify case-level predictors of IMD with DSP, and to evaluate the relationship among DSP, bacterial phenotype, and the likelihood of a fatal outcome. All IMD isolates received in Ontario between 2000 and 2006 were submitted to the Public Health Laboratories, Toronto, for confirmation of the species, serogroup determination, and susceptibility testing. Isolates were considered to be IMD strains with DSP if the penicillin MIC was ≥0.125 μg/ml. Temporal trends were evaluated using multivariable Poisson regression models. Correlates of diminished susceptibility and fatal outcome were evaluated with multivariable logistic regression models. The overall rate of IMD caused by strains with DSP in Ontario was approximately 1.20 cases per million population annually (95% confidence interval [95% CI], 0.99 to 1.46). Seventy-nine strains (21.7%) were IMD strains with DSP. There was no year-to-year trend in the incidence of IMD with DSP. IMD with DSP was strongly associated with strains of serogroups Y (odds ratio [OR], 6.3; 95% CI, 3.6 to 11.1) and W-135 (OR, 8.2; 95% CI, 4.0 to 16.7). Infection with serogroup B or C strains was associated with a marked increase in the risk of mortality (OR, 3.07; 95% CI, 1.39 to 6.75); however, no association between IMD with DSP and mortality was observed. In contrast to trends of the 1990s, the incidence of IMD with DSP was stable in Ontario between 2000 and 2006. In Ontario, the serogroup rather than the penicillin MIC is the microbiological parameter most predictive of mortality.
doi:10.1128/AAC.01077-09
PMCID: PMC2826021  PMID: 20086160
14.  Multicenter Comparison of Two Norovirus ORF2-Based Genotyping Protocols▿ †  
Journal of Clinical Microbiology  2009;47(12):3927-3932.
Point source norovirus outbreaks can be difficult to track due to high background levels of the virus in the environment and the limited strain variation in some genotyping regions. However, rapid and accurate source identification can limit the spread of a foodborne outbreak and reduce the number of cases. Harmonization of genotyping assays is critical for enabling the rapid exchange of sequence data nationally and internationally. Several regions of the genome have been proposed for this purpose, but no consensus has been reached. In the present study, two standardized genotyping protocols (region C and region D) were evaluated by nine laboratories in Canada and the United States, using a coded panel of 96 fecal specimens representing 22 different norovirus genotypes. Overall, region C typing had a success rate of 78% compared to 52% for region D; however, region D provides greater nucleotide sequence diversity for identifying new GII.4 variant strains. Significant differences in the genotyping success rate were observed among the nine participating laboratories (10% to 100%) and among the different genotypes (6% to 100%). For several genogroup II strains, reduced region D amplification correlated directly with mismatches between primer sequences and the template. Based on overall performance, we recommend the region C protocol for routine genotyping of noroviruses, while the region D protocol may be useful for identifying new GII.4 variants. Standardized genotyping protocols will enable rapid exchange of outbreak and sequence data through electronic norovirus surveillance networks.
doi:10.1128/JCM.00497-09
PMCID: PMC2786635  PMID: 19846650
15.  Association between the 2008–09 Seasonal Influenza Vaccine and Pandemic H1N1 Illness during Spring–Summer 2009: Four Observational Studies from Canada 
PLoS Medicine  2010;7(4):e1000258.
In three case-control studies and a household transmission cohort, Danuta Skowronski and colleagues find an association between prior seasonal flu vaccination and increased risk of 2009 pandemic H1N1 flu.
Background
In late spring 2009, concern was raised in Canada that prior vaccination with the 2008–09 trivalent inactivated influenza vaccine (TIV) was associated with increased risk of pandemic influenza A (H1N1) (pH1N1) illness. Several epidemiologic investigations were conducted through the summer to assess this putative association.
Methods and Findings
Studies included: (1) test-negative case-control design based on Canada's sentinel vaccine effectiveness monitoring system in British Columbia, Alberta, Ontario, and Quebec; (2) conventional case-control design using population controls in Quebec; (3) test-negative case-control design in Ontario; and (4) prospective household transmission (cohort) study in Quebec. Logistic regression was used to estimate odds ratios for TIV effect on community- or hospital-based laboratory-confirmed seasonal or pH1N1 influenza cases compared to controls with restriction, stratification, and adjustment for covariates including combinations of age, sex, comorbidity, timeliness of medical visit, prior physician visits, and/or health care worker (HCW) status. For the prospective study risk ratios were computed. Based on the sentinel study of 672 cases and 857 controls, 2008–09 TIV was associated with statistically significant protection against seasonal influenza (odds ratio 0.44, 95% CI 0.33–0.59). In contrast, estimates from the sentinel and three other observational studies, involving a total of 1,226 laboratory-confirmed pH1N1 cases and 1,505 controls, indicated that prior receipt of 2008–09 TIV was associated with increased risk of medically attended pH1N1 illness during the spring–summer 2009, with estimated risk or odds ratios ranging from 1.4 to 2.5. Risk of pH1N1 hospitalization was not further increased among vaccinated people when comparing hospitalized to community cases.
Conclusions
Prior receipt of 2008–09 TIV was associated with increased risk of medically attended pH1N1 illness during the spring–summer 2009 in Canada. The occurrence of bias (selection, information) or confounding cannot be ruled out. Further experimental and epidemiological assessment is warranted. Possible biological mechanisms and immunoepidemiologic implications are considered.
Please see later in the article for the Editors' Summary
Editors' Summary
Background
Every winter, millions of people catch influenza—a viral infection of the airways—and hundreds of thousands of people die as a result. These seasonal epidemics occur because small but frequent changes in the influenza virus mean that an immune response produced one year through infection or vaccination provides only partial protection against influenza the next year. Annual vaccination with killed influenza viruses of the major circulating strains can greatly reduce a person's risk of catching influenza. Consequently, many countries run seasonal influenza vaccination programs. In most of Canada, vaccination with a mixture of three inactivated viruses (a trivalent inactivated vaccine or TIV) is provided free to children aged 6–23 months, to elderly people, to people with long-term conditions that increase their risk of influenza-related complications, and those who provide care for them; in Ontario, free vaccination is offered to everyone older than 6 months.
In addition, influenza viruses occasionally emerge that are very different and to which human populations have virtually no immunity. These viruses can start global epidemics (pandemics) that can kill millions of people. Experts have been warning for some time that an influenza pandemic is long overdue and, in March 2009, the first cases of influenza caused by a new virus called pandemic A/H1N1 2009 (pH1N1; swine flu) occurred in Mexico. The virus spread rapidly and on 11 June 2009, the World Health Organization declared that a global pandemic of pH1N1 influenza was underway. By the end of February 2010, more than 16,000 people around the world had died from pH1N1.
Why Was This Study Done?
During an investigation of a school outbreak of pH1N1 in the late spring 2009 in Canada, investigators noted that people with illness characterized by fever and coughing had been vaccinated against seasonal influenza more often than individuals without such illness. To assess whether this association between prior vaccination with seasonal 2008–09 TIV and subsequent pH1N1 illness was evident in other settings, researchers in Canada therefore conducted additional studies using different methods. In this paper, the researchers report the results of four additional studies conducted in Canada during the summer of 2009 to assess this possible association.
What Did the Researchers Do and Find?
The researchers conducted four epidemiologic studies. Epidemiology is the study of the causes, distribution, and control of diseases in populations.
Three of the four studies were case-control studies in which the researchers assessed the frequency of prior vaccination with the 2008–09 TIV in people with pH1N1 influenza compared to the frequency among healthy members of the general population or among individuals who had an influenza-like illness but no sign of infection with an influenza virus. The researchers also did a household transmission study in which they collected information about vaccination with TIV among the additional cases of influenza that were identified in 47 households in which a case of laboratory-confirmed pH1N1 influenza had occurred. The first of the case-control studies, which was based on Canada's vaccine effectiveness monitoring system, showed that, as expected, the 2008–09 TIV provided protection against seasonal influenza. However, estimates from all four studies (which included about 1,200 laboratory-confirmed pH1N1 cases and 1,500 controls) showed that prior recipients of the 2008–09 TIV had approximately 1.4–2.5 times increased chances of developing pH1N1 illness that needed medical attention during the spring–summer of 2009 compared to people who had not received the TIV. Prior seasonal vaccination was not associated with an increase in the severity of pH1N1 illness, however. That is, it did not increase the risk of being hospitalized among those with pH1N1 illness.
What Do These Findings Mean?
Because all the investigations in this study are “observational,” the people who had been vaccinated might share another unknown characteristic that is actually responsible for increasing their risk of developing pH1N1 illness (“confounding”). Furthermore, the results reported in this study might have arisen by chance, although the consistency of results across the studies makes this unlikely. Thus, the finding of an association between prior receipt of 2008–09 TIV and an increased risk of pH1N1 illness is not conclusive and needs to be investigated further, particularly since some other observational studies conducted in other countries have reported that seasonal vaccination had no influence or may have been associated with reduced chances of pH1N1 illness. If the findings in the current study are real, however, they raise important questions about the biological interactions between seasonal and pandemic influenza strains and vaccines, and about the best way to prevent and control both types of influenza in future.
Additional Information
Please access these Web sites via the online version of this summary at http://dx.doi.org/ 10.1371/journal.pmed.1000258.
This article is further discussed in a PLoS Medicine Perspective by Cécile Viboud and Lone Simonsen
FightFlu.ca, a Canadian government Web site, provides access to information on pH1N1 influenza
The US Centers for Disease Control and Prevention provides information about influenza for patients and professionals, including specific information on H1N1 influenza
Flu.gov, a US government website, provides access to information on H1N1, avian and pandemic influenza
The World Health Organization provides information on seasonal influenza and has detailed information on pH1N1 influenza (in several languages)
The UK Health Protection Agency provides information on pandemic influenza and on pH1N1 influenza
doi:10.1371/journal.pmed.1000258
PMCID: PMC2850386  PMID: 20386731
16.  Mycobacterium tuberculosis in Ontario, Canada: Insights from IS6110 Restriction Fragment Length Polymorphism and Mycobacterial Interspersed Repetitive-Unit-Variable-Number Tandem-Repeat Genotyping▿  
Journal of Clinical Microbiology  2009;47(8):2651-2654.
A collection of 1,308 clinical Mycobacterium tuberculosis isolates from Ontario, Canada, was genotyped by IS6110 restriction fragment length polymorphism (RFLP) and mycobacterial interspersed repetitive-unit-variable-number tandem-repeat (MIRU-VNTR) analysis. RFLP or >12 MIRU-VNTR loci were necessary for resolution of Indo-Oceanic strains. The low clustering rate and high strain diversity indicate that, in Ontario, most tuberculosis results from reactivation of latent infections.
doi:10.1128/JCM.01946-08
PMCID: PMC2725669  PMID: 19494075
17.  An outbreak of vancomycin-resistant Enterococcus faecium in an acute care pediatric hospital: Lessons from environmental screening and a case-control study 
BACKGROUND
The present study describes a vancomycin-resistant enterococci (VRE) outbreak investigation and a case-control study to identify risk factors for VRE acquisition in a tertiary care pediatric hospital.
OBJECTIVE
To report an outbreak investigation and a case-control study to identify risk factors for VRE colonization or infection in hospitalized children.
METHODS
Screening for VRE cases was performed by culture or polymerase chain reaction. A case-control study of VRE-colonized patients was undertaken. Environmental screening was performed using standard culture and susceptibility methods, with pulsed-field gel electrophoresis to determine relationships between VRE isolates. Statistical analysis was performed using SAS version 9.0 (SAS Institute Inc, USA).
RESULTS
Thirty-four VRE-positive cases were identified on 10 wards between February 28, 2005, and May 27, 2005. Pulsed-field gel electrophoresis analysis confirmed a single outbreak strain that was also isolated from a video game found on one affected ward. Multivariate analysis identified cephalosporin use as the major risk factor for VRE colonization.
CONCLUSIONS
In the present study outbreak, VRE colonization was significantly associated with cephalosporin use. Because shared recreational items and environmental surfaces may be colonized by VRE, they warrant particular attention in housekeeping protocols, particularly in pediatric institutions.
PMCID: PMC2605870  PMID: 19412380
Cephalosporins; Outbreak; Pediatrics; Risk factor; VRE
18.  Of gastro and the gold standard: evaluation and policy implications of norovirus test performance for outbreak detection 
Background
The norovirus group (NVG) of caliciviruses are the etiological agents of most institutional outbreaks of gastroenteritis in North America and Europe. Identification of NVG is complicated by the non-culturable nature of this virus, and the absence of a diagnostic gold standard makes traditional evaluation of test characteristics problematic.
Methods
We evaluated 189 specimens derived from 440 acute gastroenteritis outbreaks investigated in Ontario in 2006–07. Parallel testing for NVG was performed with real-time reverse-transcriptase polymerase chain reaction (RT2-PCR), enzyme immunoassay (EIA) and electron microscopy (EM). Test characteristics (sensitivity and specificity) were estimated using latent class models and composite reference standard methods. The practical implications of test characteristics were evaluated using binomial probability models.
Results
Latent class modelling estimated sensitivities of RT2-PCR, EIA, and EM as 100%, 86%, and 17% respectively; specificities were 84%, 92%, and 100%; estimates obtained using a composite reference standard were similar. If all specimens contained norovirus, RT2-PCR or EIA would be associated with > 99.9% likelihood of at least one test being positive after three specimens tested. Testing of more than 5 true negative specimens with RT2-PCR would be associated with a greater than 50% likelihood of a false positive test.
Conclusion
Our findings support the characterization of EM as lacking sensitivity for NVG outbreaks. The high sensitivity of RT2-PCR and EIA permit identification of NVG outbreaks with testing of limited numbers of clinical specimens. Given risks of false positive test results, it is reasonable to limit the number of specimens tested when RT2-PCR or EIA are available.
doi:10.1186/1479-5876-7-23
PMCID: PMC2667494  PMID: 19323808
19.  Verification of the Combimatrix influenza detection assay for the detection of influenza A subtype during the 2007–2008 influenza season in Toronto, Canada 
Virology Journal  2009;6:37.
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.
doi:10.1186/1743-422X-6-37
PMCID: PMC2664794  PMID: 19321005
20.  Verification of the ProPneumo-1 assay for the simultaneous detection of Mycoplasma pneumoniae and Chlamydophila pneumoniae in clinical respiratory specimens 
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.
doi:10.1186/1476-0711-8-10
PMCID: PMC2655279  PMID: 19284712
21.  Characterization of culture-positive adenovirus serotypes from respiratory specimens in Toronto, Ontario, Canada: September 2007–June 2008 
Virology Journal  2009;6:11.
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.
doi:10.1186/1743-422X-6-11
PMCID: PMC2656483  PMID: 19171030
22.  The relative test performance characteristics of two commercial assays for the detection of Mycobacterium tuberculosis complex in paraffin-fixed human biopsy specimens 
Diagnostic Pathology  2008;3:37.
The Seeplex™ TB Detection-2 assay (Rockville, MD) is a nested endpoint PCR for the Mycobacterium tuberculosis complex (MTBC) targets IS6110 and MPB64 that utilizes dual priming oligonucleotide technology. When used to detect the presence of MTBC DNA in formalin-fixed paraffin-embedded tissue specimens, the sensitivity and specificity of this assay is equivalent to a labor-intensive traditional endpoint PCR assay and is more sensitive than a commercial real-time PCR assay.
doi:10.1186/1746-1596-3-37
PMCID: PMC2546368  PMID: 18778465
24.  Comparison of Two Versions of the IDI-MRSA Assay Using Charcoal Swabs for Prospective Nasal and Nonnasal Surveillance Samples▿  
Journal of Clinical Microbiology  2007;45(7):2278-2280.
An updated IDI-MRSA assay version was released to address the assay's 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.
doi:10.1128/JCM.00469-07
PMCID: PMC1932969  PMID: 17522279
25.  Verification of the IDI-MRSA Assay for Detecting Methicillin-Resistant Staphylococcus aureus in Diverse Specimen Types in a Core Clinical Laboratory Setting 
Journal of Clinical Microbiology  2006;44(10):3794-3796.
The IDI-MRSA assay has a sensitivity of 96% and a specificity of 96% when used to screen patients at extranasal sites. This verification study used previously unverified swabs and was undertaken in a core medical laboratory using nonmicrobiology technologists trained in sample processing, molecular laboratory work flow, and PCR practice.
doi:10.1128/JCM.01509-06
PMCID: PMC1594777  PMID: 17021113

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