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1.
Can J Microbiol ; : 1-12, 2019 Oct 29.
Artigo em Inglês | MEDLINE | ID: mdl-31661630

RESUMO

This study examined the phylogenetic structure of serotype a Haemophilus influenzae (Hia) isolates recovered from patients in Canada. Hia isolates from 490 separate patients and an American Type Culture Collection (ATCC) strain were analyzed by multilocus sequence typing (MLST), with 18 different sequence types (STs) identified. Most (85.7%) Hia patient isolates were typed as ST-23 and another 12.7% belonged to 14 different STs with 6, 5, or 4 MLST gene loci related to ST-23 (ST-23 complex). Core genome single-nucleotide variation phylogeny (SNVPhyl) on whole genome sequence (WGS) data of 121 Hia patient isolates representing all identified STs and the ATCC strain revealed 2 phylogenetic populations, with all the ST-23 complex isolates within 1 population. The other phylogenetic population contained only the ATCC strain and 3 patient isolates. Concatenated hitABC sequences retrieved from WGS data and analyzed by MEGA (Molecular Evolutionary Genetic Analysis) alignment confirmed the phylogeny obtained by SNVPhyl. The sodC gene was found only in isolates in the minor phylogenetic population. The 2 phylogenetic populations of the Canadian Hia isolates are similar to the 2 clonal divisions described for serotype b H. influenzae. Combining MLST, core SNVPhyl, and hitABC gene sequence alignment showed that most (99.4%) Canadian Hia patient isolates belonged to 1 major phylogenetic population.

2.
J Infect Dis ; 2019 Sep 24.
Artigo em Inglês | MEDLINE | ID: mdl-31549165

RESUMO

BACKGROUND: Annual influenza immunization is recommended for people with chronic obstructive pulmonary disease (COPD) by all major COPD clinical practice guidelines. We sought to determine the seasonal influenza vaccine effectiveness (VE) against laboratory-confirmed influenza-associated hospitalizations among older adults with COPD. METHODS: We conducted a test-negative study of influenza VE in community-dwelling older adults with COPD in Ontario, Canada using health administrative data and respiratory specimens collected from patients tested for influenza during the 2010-11 to 2015-16 influenza seasons. Influenza vaccination was ascertained from physician and pharmacist billing claims. Multivariable logistic regression was used to estimate the adjusted odds ratio of influenza vaccination in people with, compared to those without, laboratory-confirmed influenza. RESULTS: Receipt of seasonal influenza vaccine was associated with an adjusted 22% (95% confidence interval [CI], 15%-27%) reduction in laboratory-confirmed influenza-associated hospitalization. Adjustment for potential misclassification of vaccination status increased this to 43% (95% CI, 35%-52%). Vaccine effectiveness was not found to vary by patient- or influenza-related variables. CONCLUSIONS: During the studied influenza seasons, influenza vaccination was at least modestly effective in reducing laboratory-confirmed influenza-associated hospitalizations in people with COPD. The imperfect effectiveness emphasizes the need for better influenza vaccines and other preventive strategies.

3.
J Clin Oncol ; 37(30): 2795-2804, 2019 Oct 20.
Artigo em Inglês | MEDLINE | ID: mdl-31465264

RESUMO

PURPOSE: Seasonal influenza vaccination is recommended for patients with cancer despite concerns of disease or treatment-associated immunosuppression. The objective of this study was to evaluate vaccine effectiveness (VE) against laboratory-confirmed influenza for patients with cancer. PATIENTS AND METHODS: We conducted an observational test-negative design study of previously diagnosed patients with cancer 18 years of age and older who underwent influenza testing during the 2010-2011 to 2015-2016 influenza seasons in Ontario, Canada. We linked individual-level cancer registry, respiratory virus testing, and health administrative data to identify the study population and outcomes. Vaccination status was determined from physician and pharmacist billing claims. We used multivariable logistic regression to estimate VE, adjusting for age, sex, rurality, income quintile, cancer characteristics, chemotherapy exposure, comorbidities, previous health care use, influenza season, and calendar time. RESULTS: We identified 26,463 patients with cancer who underwent influenza testing, with 4,320 test-positive cases (16%) and 11,783 (45%) vaccinated. Mean age was 70 years, 52% were male, mean time since diagnosis was 6 years, 69% had solid tumor malignancies, and 23% received active chemotherapy. VE against laboratory-confirmed influenza was 21% (95% CI, 15% to 26%), and VE against laboratory-confirmed influenza hospitalization was 20% (95% CI, 13% to 26%). For patients with solid tumor malignancies, VE was 25% (95% CI, 18% to 31%), compared with 8% (95% CI, -5% to 19%) for patients with hematologic malignancies (P = .015). Active chemotherapy usage did not significantly affect VE, especially among patients with solid tumor cancer. CONCLUSION: Our results support recommendations for influenza vaccination for patients with cancer. VE was decreased for patients with hematologic malignancies, and there was no significant difference in VE among patients with solid tumor cancer receiving active chemotherapy. Strategies to optimize influenza prevention among patients with cancer are warranted.

4.
Vaccine ; 37(31): 4392-4400, 2019 Jul 18.
Artigo em Inglês | MEDLINE | ID: mdl-31221563

RESUMO

BACKGROUND: Linking data on laboratory specimens collected during clinical practice with health administrative data permits highly powered vaccine effectiveness (VE) studies to be conducted at relatively low cost, but bias from using convenience samples is a concern. We evaluated the validity of using such data for estimating VE. METHODS: We created the Flu and Other Respiratory Viruses Research (FOREVER) Cohort by linking individual-level data on respiratory virus laboratory tests, hospitalizations, emergency department visits, and physician services. For community-dwelling adults aged > 65 years, we assessed the presence and magnitude of information and selection biases, generated VE estimates under various conditions, and compared our VE estimates with those from other studies. RESULTS: We included 65,648 unique testing episodes obtained from 54,434 individuals during the 2010-11 to 2015-16 influenza seasons. To examine information bias, we found the proportion testing positive for influenza for patients with unknown interval from illness onset to specimen collection was more similar to patients for whom illness onset date was ≤ 7 days before specimen collection than to patients for whom illness onset was > 7 days before specimen collection. To assess the presence of selection bias, we found the likelihood of influenza testing was comparable between vaccinated and unvaccinated individuals, although the adjusted odds ratios were significantly greater than 1 for some healthcare settings and during some influenza seasons. Over 6 seasons, VE estimates ranged between 36% (95%CI, 27-44%) in 2010-11 and 5% (95%CI, -2, 11%) in 2014-15. VE estimates were similar under a range of conditions, but were consistently higher when accounting for misclassification of vaccination status through a quantitative sensitivity analysis. VE estimates from the FOREVER Cohort were comparable to those from other studies. CONCLUSIONS: Routinely collected laboratory and health administrative data contained in the FOREVER Cohort can be used to estimate influenza VE in community-dwelling older adults.

5.
Pediatr Infect Dis J ; 38(4): 362-369, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30882725

RESUMO

BACKGROUND: Respiratory illnesses are a major contributor to pediatric hospitalizations, with influenza and respiratory syncytial virus (RSV) causing substantial morbidity and cost each season. We compared the characteristics and outcomes of children 0-59 months of age who were hospitalized with laboratory-confirmed influenza or RSV between 2009 and 2014 in Ontario, Canada. METHODS: We included hospitalized children who were tested for influenza A, influenza B and RSV and were positive for a single virus. We characterized individuals by their demographics and healthcare utilization patterns and compared their hospital outcomes, in-hospital cost and postdischarge healthcare use by virus type and by presence of underlying comorbidities. RESULTS: We identified and analyzed 7659 hospitalizations during which a specimen tested positive for influenza or RSV. Children with RSV were the youngest whereas children with influenza B were the oldest [median ages 6 months (interquartile range: 2-17 months) and 25 months (interquartile range: 10-45 months), respectively]. Complex chronic conditions were more prevalent among children with all influenza (sub)types than RSV (31%-34% versus 20%). In-hospital outcomes were similar by virus type, but in children with comorbidities, postdischarge outcomes varied. We observed no differences in in-hospital cost between viruses or by presence of comorbidities [overall median cost: $4150 Canadian dollars (interquartile range: $3710-$4948)]. CONCLUSIONS: Influenza and RSV account for large numbers of pediatric hospitalizations. RSV and influenza were similar in terms of severity and cost in hospitalized children. Influenza vaccination should be promoted in pregnant women and young children, and a vaccine against RSV would mitigate the high burden of RSV.

6.
Pediatr Infect Dis J ; 2018 Aug 27.
Artigo em Inglês | MEDLINE | ID: mdl-30153230

RESUMO

BACKGROUND: Respiratory illnesses are a major contributor to pediatric hospitalizations, with influenza and respiratory syncytial virus (RSV) causing substantial morbidity and cost each season. We compared the characteristics and outcomes of children 0-59 months of age who were hospitalized with laboratory-confirmed influenza or RSV between 2009 and 2014 in Ontario, Canada. METHODS: We included hospitalized children who were tested for influenza A, influenza B, and RSV, and were positive for a single virus. We characterized individuals by their demographics and healthcare utilization patterns, and compared their hospital outcomes, in-hospital cost, and post-discharge healthcare use by virus type and by presence of underlying comorbidities. RESULTS: We identified and analyzed 7,659 hospitalizations during which a specimen tested positive for influenza or RSV. Children with RSV were the youngest whereas children with influenza B were the oldest (median ages 6 months [interquartile range {IQR}: 2-17 months] and 25 months [IQR: 10-45 months], respectively). Complex chronic conditions were more prevalent among children with all influenza (sub)types than RSV (31%-34% vs 20%). In-hospital outcomes were similar by virus type, but in children with comorbidities, post-discharge outcomes varied. We observed no differences in in-hospital cost between viruses or by presence of comorbidities (overall median cost: $4,150 CAD [IQR: $3,710-$4,948]). CONCLUSION: Influenza and RSV account for large numbers of pediatric hospitalizations. RSV and influenza were similar in terms of severity and cost in hospitalized children. Influenza vaccination should be promoted in pregnant women and young children, and a vaccine against RSV would mitigate the high burden of RSV.

7.
J Antimicrob Chemother ; 73(suppl_7): vii20-vii31, 2018 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-29982573

RESUMO

Objectives: This study characterized the 11 most predominant serotypes of invasive Streptococcus pneumoniae infections collected by the annual SAVE study in Canada, between 2011 and 2015. Methods: A subset of the 11 most predominant serotypes (7F, 19A, 22F, 3, 12F, 11A, 9N, 8, 33F, 15A and 6C) collected by the SAVE study was analysed using PFGE and MLST, as well as PCR to identify pilus-encoding genes. WGS analyses were performed on a subset of the above isolates plus a random selection of background strains. Results: Of the predominant serotypes analysed, 7F, 33F and 19A were obtained more commonly from children <6 years of age, whereas 15A, 6C, 22F and 11A were more common in adults >65 years of age. Pneumococcal pilus PI-1 was identified in antimicrobial-susceptible serotype 15A (61/212) and <10% of 6C isolates (16/188). PI-2 was found in serotype 7F (683/701) and two-thirds of 11A isolates (162/241). Only serotype 19A-ST320 possessed both pili. Molecular and phylogenetic analyses identified serotypes 19A, 15A, 6C, 9N and 33F as highly diverse, whereas 7F, 22F and 11A demonstrated clonality. Antimicrobial resistance determinants were common within diverse serotypes, and usually similar within a clonal complex. Conclusions: Despite successful use of conjugate vaccines, S. pneumoniae remains a highly diverse organism in Canada. Several predominant serotypes, both antimicrobial susceptible and MDR, have demonstrated rapid clonal expansion or an increase in diversity. As S. pneumoniae continues to evolve in Canada, WGS will be a necessary component in the ongoing surveillance of antimicrobial-resistant and expanding clones.

8.
Vaccine ; 36(31): 4701-4707, 2018 07 25.
Artigo em Inglês | MEDLINE | ID: mdl-29937245

RESUMO

The 13-valent conjugate vaccine (PCV13) was recommended for childhood immunization programs in 2010 in Canada and has decreased the incidence of invasive pneumococcal disease (IPD) in children and changed the epidemiology of IPD in adults. This study investigated the epidemiology of IPD in adults 65 years of age and older in Canada. A total of 7282 invasive S. pneumoniae isolated from adults ≥65 years old were serotyped from 2010 to 2016 and antimicrobial susceptibility was performed on 2527 isolates. Serotyping was performed by Quellung reaction using commercial antisera and antimicrobial susceptibilities were determined by broth microdilution. PCV7 serotypes decreased non-significantly from 2010 to 2016 from 9.1% (n = 96) to 6.7% (n = 72) while the additional six PCV13 serotypes declined significantly from 39.5% (n = 418) to 18.6% (n = 201) (p < 0.05). The 23-valent pneumococcal polysaccharide vaccine (PPV23) and non-vaccine (NVT) serotypes increased from 26.3% (n = 278) to 36.2% (n = 393) (p < 0.05), and from 25.1% (n = 266) to 38.4% (n = 416) (p < 0.05), respectively. There were no significant changes in antimicrobial resistance rates from 2011 to 2016: 24.1% of the IPD from adults ≥65 years were resistant to clarithromycin (n = 609), 10.0% to doxycycline (n = 254), 11.8% to penicillin (n = 299), 5.2% to cefuroxime (n = 131), 6.6% to clindamycin (n = 168), 6.0% to trimethoprim-sulfamethoxazole (n = 152), and 0.5% (n = 12) to ceftriaxone. Although overall incidence of IPD in adults ≥65 years has remained relatively constant from 2010 to 2016, childhood PCV13 vaccination programs have been successful in indirectly reducing IPD caused by PCV13 serotypes in adults through herd immunity effects.


Assuntos
Infecções Pneumocócicas/epidemiologia , Infecções Pneumocócicas/microbiologia , Vacinas Pneumocócicas/administração & dosagem , Sorogrupo , Streptococcus pneumoniae/classificação , Streptococcus pneumoniae/isolamento & purificação , Idoso , Idoso de 80 Anos ou mais , Antibacterianos/farmacologia , Canadá/epidemiologia , Farmacorresistência Bacteriana , Feminino , Humanos , Imunidade Coletiva , Incidência , Masculino , Testes de Sensibilidade Microbiana , Sorotipagem
9.
N Engl J Med ; 378(4): 345-353, 2018 Jan 25.
Artigo em Inglês | MEDLINE | ID: mdl-29365305

RESUMO

BACKGROUND: Acute myocardial infarction can be triggered by acute respiratory infections. Previous studies have suggested an association between influenza and acute myocardial infarction, but those studies used nonspecific measures of influenza infection or study designs that were susceptible to bias. We evaluated the association between laboratory-confirmed influenza infection and acute myocardial infarction. METHODS: We used the self-controlled case-series design to evaluate the association between laboratory-confirmed influenza infection and hospitalization for acute myocardial infarction. We used various high-specificity laboratory methods to confirm influenza infection in respiratory specimens, and we ascertained hospitalization for acute myocardial infarction from administrative data. We defined the "risk interval" as the first 7 days after respiratory specimen collection and the "control interval" as 1 year before and 1 year after the risk interval. RESULTS: We identified 364 hospitalizations for acute myocardial infarction that occurred within 1 year before and 1 year after a positive test result for influenza. Of these, 20 (20.0 admissions per week) occurred during the risk interval and 344 (3.3 admissions per week) occurred during the control interval. The incidence ratio of an admission for acute myocardial infarction during the risk interval as compared with the control interval was 6.05 (95% confidence interval [CI], 3.86 to 9.50). No increased incidence was observed after day 7. Incidence ratios for acute myocardial infarction within 7 days after detection of influenza B, influenza A, respiratory syncytial virus, and other viruses were 10.11 (95% CI, 4.37 to 23.38), 5.17 (95% CI, 3.02 to 8.84), 3.51 (95% CI, 1.11 to 11.12), and 2.77 (95% CI, 1.23 to 6.24), respectively. CONCLUSIONS: We found a significant association between respiratory infections, especially influenza, and acute myocardial infarction. (Funded by the Canadian Institutes of Health Research and others.).


Assuntos
Hospitalização/estatística & dados numéricos , Influenza Humana/complicações , Infarto do Miocárdio/etiologia , Idoso , Idoso de 80 Anos ou mais , Feminino , Humanos , Incidência , Vírus da Influenza A/isolamento & purificação , Vírus da Influenza B/isolamento & purificação , Influenza Humana/diagnóstico , Masculino , Pessoa de Meia-Idade , Infarto do Miocárdio/epidemiologia , Ontário/epidemiologia , Risco
10.
PLoS One ; 12(11): e0187834, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-29149183

RESUMO

Uncertainty remains regarding the magnitude of effectiveness of influenza vaccines for preventing serious outcomes, especially among young children. We estimated vaccine effectiveness (VE) against laboratory-confirmed influenza hospitalizations among children aged 6-59 months. We used the test-negative design in hospitalized children in Ontario, Canada during the 2010-11 to 2013-14 influenza seasons. We used logistic regression models adjusted for age, season, and time within season to calculate VE estimates by vaccination status (full vs. partial), age group, and influenza season. We also assessed VE incorporating prior history of influenza vaccination. We included specimens from 9,982 patient hospitalization episodes over four seasons, with 12.8% testing positive for influenza. We observed variation in VE by vaccination status, age group, and influenza season. For the four seasons combined, VE was 60% (95%CI, 44%-72%) for full vaccination and 39% (95%CI, 17%-56%) for partial vaccination. VE for full vaccination was 67% (95%CI, 48%-79%) for children aged 24-59 months, 48% (95%CI, 12%-69%) for children aged 6-23 months, 77% (95%CI, 47%-90%) for 2010-11, 59% (95%CI, 13%-81%) for 2011-12, 33% (95%CI, -18% to 62%) for 2012-13, and 72% (95%CI, 42%-86%) for 2013-14. VE in children aged 24-59 months appeared similar between those vaccinated in both the current and previous seasons and those vaccinated in the current season only, with the exception of 2012-13, when VE was lower for those vaccinated in the current season only. Influenza vaccination is effective in preventing pediatric laboratory-confirmed influenza hospitalizations during most seasons.


Assuntos
Vacinas contra Influenza/administração & dosagem , Influenza Humana/prevenção & controle , Pré-Escolar , Feminino , Humanos , Lactente , Masculino , Ontário
11.
J Virol Methods ; 248: 39-43, 2017 10.
Artigo em Inglês | MEDLINE | ID: mdl-28633962

RESUMO

BACKGROUND: Timely identification of respiratory virus infection is essential to mitigate inappropriate antibiotic use and to implement appropriate treatment and/or infection control procedures. As such, multiplexed PCR assays have become standard in many virology laboratories. OBJECTIVES: To compare the Seeplex RV15 (test of record) with two newer generation multiplex assays, the Anyplex II RV16 and the xTAG respiratory virus panels. STUDY DESIGN: Two hundred and three retrospective and 36 prospective respiratory samples were tested by all three assays. Samples were deemed to be positive if they tested positive for a virus by at least two of the three respective assays. Negative samples also had to test negative by at least two of the three assays. Inconclusive samples were those that showed band signal intensity between 0 and 100 on the RV15, but had not been previously tested on the RV16 or xTAG. RESULTS AND CONCLUSIONS: Overall sensitivity and specificity of all three assays were similar (∼85% and 100%, respectively). Given each assay can identify multiple different viruses, the targets reported by one assay did not always agree with each target from another assay. Partial discordant rates were 47% and 21% for positive and negative samples, respectively. These higher than expected partial discordant rates may be due to primer or chemistry differences amongst the three multiplex assays.


Assuntos
Reação em Cadeia da Polimerase Multiplex/métodos , Infecções Respiratórias/diagnóstico , Infecções Respiratórias/virologia , Viroses/diagnóstico , Vírus/isolamento & purificação , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Líquido da Lavagem Broncoalveolar/virologia , Criança , Pré-Escolar , Feminino , Humanos , Lactente , Recém-Nascido , Masculino , Pessoa de Meia-Idade , Nasofaringe/virologia , Estudos Prospectivos , Kit de Reagentes para Diagnóstico , Estudos Retrospectivos , Sensibilidade e Especificidade , Viroses/virologia , Vírus/genética , Adulto Jovem
12.
Can J Infect Dis Med Microbiol ; 25(4): 207-10, 2014 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-25285125

RESUMO

BACKGROUND: Disease monitoring of viruses using real-time polymerase chain reaction (PCR) requires knowledge of the precision of the test to determine what constitutes a significant change. Calculation of quantitative PCR confidence limits requires bivariate statistical methods. OBJECTIVE: To develop a simple-to-use graphical user interface to determine the uncertainty of measurement (UOM) of BK virus, cytomegalovirus (CMV) and Epstein-Barr virus (EBV) real-time PCR assays. METHODS: Thirty positive clinical samples for each of the three viral assays were repeated once. A graphical user interface was developed using a spreadsheet (Excel, Microsoft Corporation, USA) to enable data entry and calculation of the UOM (according to Fieller's theorem) and PCR efficiency. RESULTS: The confidence limits for the BK virus, CMV and EBV tests were ∼0.5 log, 0.5 log to 1.0 log, and 0.5 log to 1.0 log, respectively. The efficiencies of these assays, in the same order were 105%, 119% and 90%. The confidence limits remained stable over the linear range of all three tests. DISCUSSION: A >5 fold (0.7 log) and a >3-fold (0.5 log) change in viral load were significant for CMV and EBV when the results were ≤1000 copies/mL and >1000 copies/mL, respectively. A >3-fold (0.5 log) change in viral load was significant for BK virus over its entire linear range. PCR efficiency was ideal for BK virus and EBV but not CMV. Standardized international reference materials and shared reporting of UOM among laboratories are required for the development of treatment guidelines for BK virus, CMV and EBV in the context of changes in viral load.

13.
J Acquir Immune Defic Syndr ; 64(5): 443-7, 2013 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-24231785

RESUMO

OBJECTIVE: To examine whether baseline clinical genotypes are equivalent to diagnostic serum genotypes for surveillance of HIV transmitted drug resistance (TDR). DESIGN: Current HIV TDR surveillance in Canada is conducted through genotyping remnant diagnostic sera from new HIV diagnoses. As part of routine care, baseline genotyping is now conducted on all newly diagnosed HIV infections, with TDR data being generated a second time on the same patients. METHODS: Surveillance genotyping, on HIV diagnostic serum, was performed on newly diagnosed HIV cases from 2007 to 2010 in Alberta, Canada. All subjects with a baseline clinical genotype result on file, and no evidence of antiretroviral therapy, were studied further. The HIV sequences from diagnosis and from the first clinical genotype were compared according to elapsed time between testing and by evaluating timing of infection based on BED capture enzyme immunoassay (BED-CEIA, abbreviated as BED in this article). RESULTS: Eighty-seven genotype pairs were available for analysis, most of which were subtype B. The time between genotypes ranged from 0 to 755 days, with a median of 36 days and an interquartile range of 155.25 days. Genetic distance between genotypes varied between 0 and 0.03389 substitutions per site and did not correlate with sampling times. There was a tendency for the genotypes of infections classified as recent by BED to be more similar to their clinical genotypes but this effect was lost when adjusted for elapsed time between tests. There was no difference in the identified drug resistance. CONCLUSIONS: Baseline clinical genotypes from treatment-naive patients may be used for HIV TDR surveillance.


Assuntos
Farmacorresistência Viral , Infecções por HIV/transmissão , Infecções por HIV/virologia , HIV/genética , HIV/isolamento & purificação , Alberta , Monitoramento Epidemiológico , Genótipo , Humanos
14.
Lab Chip ; 13(20): 4087-95, 2013 Oct 21.
Artigo em Inglês | MEDLINE | ID: mdl-23966212

RESUMO

For effective clinical uptake of the lab on a chip/point of care technology (LOC-POC), in addition to cost advantages LOC-POC devices should offer multiple patient screening panels for related diseases as well as cold-chain transportation and storage abilities. We recently described a device that performs polymerase chain reaction (PCR) to simultaneously screen raw clinical samples from up to 16 patients for multiple infectious agents (Manage et al., Lab Chip, 2013, 13, 2576-2584). This cassette contains glass capillaries with desiccated semi-solid acrylamide gels that include all the reagents except for the sample, with integrated quality control. Here we report the development of protocols to store assembled PCR cassettes at room temperature, 4 °C or -20 °C as well as at +40 °C. We show that our cassettes are stable, with no loss of activity for at least 3 months at RT and at least 7 months at 4 °C and -20 °C. However, the activity of desiccated cassettes degrades when stored for more than 2 weeks at 40 °C, insufficient time for post-manufacture delivery and use of cassette PCR. To address this, we have evaluated two stage storage protocols. PCR cassettes can initially be stored at 4 °C and -20 °C for prolonged periods of time and removed for shorter term storage at RT, retaining activity for at least a month, which would facilitate transport to remote areas for testing. Effective use of cassette PCR in high temperature regions of the world, for experimental purposes defined here as 40 °C, appears to be feasible only after a first stage storage in the cold, followed by no more than 1 week at 40 °C. This should allow sufficient time for delivery by the manufacturer to a central area well served by power and refrigeration, for later ambient temperature transport and use in under-resourced areas that lack refrigeration.


Assuntos
Acrilamidas/química , Sistemas Automatizados de Assistência Junto ao Leito , Reação em Cadeia da Polimerase/instrumentação , Géis , Vidro/química , Herpesvirus Humano 1/genética , Herpesvirus Humano 1/isolamento & purificação , Herpesvirus Humano 2/genética , Herpesvirus Humano 2/isolamento & purificação , Humanos , Temperatura Ambiente , Fatores de Tempo
15.
Lab Chip ; 13(13): 2576-84, 2013 Jul 07.
Artigo em Inglês | MEDLINE | ID: mdl-23471315

RESUMO

This work describes a self-contained, simple, disposable, and inexpensive gel capillary cassette for DNA amplification in near point of care settings. The cassette avoids the need for pumps or valves during raw sample delivery or polymerase chain reaction (PCR) amplification steps. The cassette contains capillary reaction units that can be stored at room temperature for up to 3 months. The current cassette configuration format simultaneously tests up to 16 patients for two or more targets, accommodates different sample types on the same cassette, has integrated positive and negative controls and allows flexibility for multiple geometries. PCR reagents in the cassette are desiccated to allow storage at room temperature with rehydration by raw sample at the time of testing. The sample is introduced to the cassette via a transfer pipette simply by capillary force. DNA amplification was carried out in a portable prototype instrument for PCR thermal cycling with fluorescence detection of amplified products by melt curve analysis (MCA). To demonstrate performance, raw genital swabs and urine were introduced to the same cassette to simultaneously detect four sexually transmitted infections. Herpes Simplex Viruses (HSV-1 and HSV-2) were detected from raw genital swabs. Ureaplasma urealyticum (UU) and Mycoplasma homonis (MH) were detected from raw urine. Results for multiple patients were obtained in as little as 50 min. This platform allows multiparameter clinical testing with a pre-assembled cassette that requires only the introduction of raw sample. Modification of the prototype device to accommodate larger cassettes will ultimately provide high throughput simultaneous testing of even larger numbers of samples for many different targets, as is required for some clinical applications. Combinations of wax and/or polymer cassettes holding capillary reaction units are feasible. The components of the cassette are suited to mass production and robotic assembly to produce a readily manufactured disposable reaction cassette that can be configured for disease-specific testing panels. Rapid testing with a disposable reaction cassette on an inexpensive instrument will enable on the spot evaluation of patients in the clinic for faster medical decision-making and more informed therapeutic choices.


Assuntos
DNA Bacteriano/urina , DNA Viral/análise , Reação em Cadeia da Polimerase/métodos , Feminino , Géis/química , Herpesvirus Humano 1/genética , Herpesvirus Humano 2/genética , Humanos , Mycoplasma hominis/genética , Reação em Cadeia da Polimerase/instrumentação , Temperatura Ambiente , Ureaplasma urealyticum/genética , Vagina/virologia
16.
J Clin Microbiol ; 51(2): 701-4, 2013 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-23224082

RESUMO

We describe an immunocompromised patient who developed a large frontal brain abscess caused by Legionella micdadei. This is, to our knowledge, a rare case of culture-proven Legionella central nervous system infection.


Assuntos
Abscesso Encefálico/microbiologia , Legionella/genética , Legionelose/microbiologia , Autopsia , Encéfalo/patologia , Abscesso Encefálico/diagnóstico , Evolução Fatal , Humanos , Hospedeiro Imunocomprometido , Legionella/classificação , Legionelose/diagnóstico , Imagem por Ressonância Magnética , Masculino , Pessoa de Meia-Idade , RNA Ribossômico 16S
18.
Lab Chip ; 12(9): 1664-71, 2012 May 07.
Artigo em Inglês | MEDLINE | ID: mdl-22426784

RESUMO

Herpes simplex virus (HSV) is one of the most prevalent viruses, with acute and recurrent infections in humans. The current gold standard for the diagnosis of HSV is viral culture which takes 2-14 days and has low sensitivity. In contrast, DNA amplification by polymerase chain reaction (PCR) can be performed within 1-2 h. We here describe a multiparameter PCR assay to simultaneously detect HSV-1 and HSV-2 DNA templates, together with integrated positive and negative controls, with product detection by melting curve analysis (MCA), in an array of semi-solid polyacrylamide gel posts. Each gel post is 0.67 µL in volume, and polymerized with all the components required for PCR. Both PCR and MCA can currently be performed in one hour and 20 min. Unprocessed genital swabs collected in universal transport medium were directly added to the reagents before or after polymerization, diffusing from atop the gel posts. The gel post platform detects HSV templates in as little as 2.5 nL of raw sample. In this study, 45 genital swab specimens were tested blindly as a preliminary validation of this platform. The concordance of PCR on gel posts with conventional PCR was 91%. The primer sequestration method introduced here (wherein different primers are placed in different sets of posts) enables the simultaneous detection of multiple pathogens for the same sample, together with positive and negative controls, on a single chip. This platform accepts unprocessed samples and is readily adaptable to detection of multiple different pathogens or biomarkers for point-of-care diagnostics.


Assuntos
DNA Viral/análise , Genitália/virologia , Herpesvirus Humano 1/isolamento & purificação , Herpesvirus Humano 2/isolamento & purificação , Reação em Cadeia da Polimerase/métodos , Primers do DNA , Desenho de Equipamento , Herpes Genital/diagnóstico , Herpes Genital/virologia , Herpesvirus Humano 1/genética , Herpesvirus Humano 2/genética , Humanos , Limite de Detecção , Desnaturação de Ácido Nucleico , Reação em Cadeia da Polimerase/instrumentação , Projetos de Pesquisa , Espectrometria de Fluorescência , Temperatura Ambiente
19.
J Clin Microbiol ; 50(1): 25-9, 2012 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-22090403

RESUMO

Clinical cervical cytology specimens (n = 466) collected in PreservCyt (Hologic Inc.) were used to evaluate the agreement between Hybrid Capture 2 (hc2; Qiagen) and cobas 4800 (c4800; Roche Molecular Diagnostics) for the detection of high-risk human papillomavirus (HR HPV) genotype infections. The agreement between the two assays was 93.8% (kappa = 0.87; 95% confidence interval, 0.828 to 0.918), with 186 and 251 concordant positive and negative results, respectively. All 186 concordant positives were confirmed using the Linear Array (LA; Roche Molecular Diagnostics) genotyping test. Of the 29 samples with discordant results (6.2%), 18 were hc2 positive and LA verified 17 as positive for HR HPV. Eleven discordant specimens were c4800 positive, and LA confirmed 5 as positive for HR HPV. As of October 2009, practice guidelines in Alberta, Canada, recommend reflex HPV testing for women over 30 years old with atypical squamous cells of undetermined significance (ASCUS) and for women over 50 years old with low-grade squamous intraepithelial lesions (LSIL) to help prioritize those who should undergo further evaluation. In this study, agreement between hc2 and c4800 results for samples from women over 30 years old with ASCUS cytology was 92.3% (n = 13), while no samples from women over 50 years old with LSIL cytology were identified for analysis.


Assuntos
Técnicas de Diagnóstico Molecular/métodos , Papillomaviridae/isolamento & purificação , Infecções por Papillomavirus/diagnóstico , Manejo de Espécimes/métodos , Virologia/métodos , Adolescente , Adulto , Alberta , Meios de Cultura/química , DNA Viral/genética , DNA Viral/isolamento & purificação , Feminino , Genótipo , Humanos , Pessoa de Meia-Idade , Papillomaviridae/classificação , Guias de Prática Clínica como Assunto , Adulto Jovem
20.
Sex Transm Dis ; 37(11): 696-9, 2010 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-20693937

RESUMO

BACKGROUND: Herpes simplex virus (HSV)-type specific serology (TSS) testing has been commercially available for nearly a decade. Guidelines on appropriate use of such testing exist, including Canadian-based recommendations. Despite this, most Canadian laboratories do not offer HSV-type specific serology and many provide only nontype-specific HSV serology tests. METHODS: At the Alberta Provincial Laboratory, HSV TSS is performed using the following algorithm (termed the Alberta algorithm). Eligible specimens are first tested with a nontype-specific kit (Behring Enzygnost IgG). If positive, sera are then tested using the Focus HerpeSelect-2 assay to establish HSV-2 infection. If the HerpeSelect-2 result is negative, the result is reported as anti-HSV-1 positive. In this study we sought a validation of the Alberta algorithm by testing 344 serum samples with the Behring Enzygnost IgG assay, the type specific Focus HerpeSelect 1 and 2 assays, and by Western blot (WB). RESULTS: Taking the WB as a gold standard, the Behring Enzygnost IgG assay showed a sensitivity of 94% and a specificity of 100%, whereas the Alberta algorithm had a sensitivity of 92% and a specificity of 97% for the detection of HSV-2 antibodies, and a sensitivity of 94% and a specificity of 100% for the detection of HSV-1 antibodies in HSV-2 negative sera. Focus HerpeSelect 1 and HerpeSelect 2 sensitivities against WB were 88% and 91%, whereas specificities were 95% and 97% for HSV-1 and HSV-2, respectively. CONCLUSIONS: The Alberta algorithm was at least equivalent to HerpeSelect 1 and 2 in detecting anti-HSV antibodies. Although sensitivity and specificity were higher, the differences were not statistically significant.


Assuntos
Algoritmos , Anticorpos Antivirais/sangue , Herpes Genital/diagnóstico , Herpes Simples/diagnóstico , Herpesvirus Humano 1/imunologia , Herpesvirus Humano 2/imunologia , Kit de Reagentes para Diagnóstico , Alberta , Anticorpos Antivirais/imunologia , Ensaio de Imunoadsorção Enzimática , Herpes Genital/imunologia , Herpes Genital/virologia , Herpes Simples/imunologia , Herpes Simples/virologia , Humanos , Imunoglobulina G/sangue , Sensibilidade e Especificidade
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