Shelly Bolotin

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

Abstract 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.

A new sentinel surveillance system for severe influenza in England shows a shift in age distribution of hospitalised cases in the post-pandemic period.

Background The World Health Organization and European Centre for Disease Prevention and Control have highlighted the importance of establishing systems to monitor severe influenza. Following the H1N1 (2009) influenza pandemic, a sentinel network of 23 Trusts, the UK Severe Influenza Surveillance System (USISS), was established to monitor hospitalisations due to confirmed seasonal influenza in England. This article presents the results of the first season of operation of USISS in 2010/11. Methodology/Principal Findings A case was defined as a person hospitalised with confirmed influenza of any type. Weekly aggregate numbers of hospitalised influenza cases, broken down by flu type and level of care, were submitted by participating Trusts. Cases in 2010/11 were compared to cases during the 2009 pandemic in hospitals with available surveillance data for both time periods (n = 19). An unexpected resurgence in seasonal A/H1N1 (2009) influenza activity in England was observed in December 2010 with reports of severe disease. Reported cases over the period of 4 October 2010 to 13 February 2011 were mostly due to influenza A/H1N1 (2009). One thousand and seventy-one cases of influenza A/H1N1 (2009) occurred over this period compared to 409 at the same Trusts over the 2009/10 pandemic period (1 April 2009 to 6 January 2010). Median age of influenza A/H1N1 (2009) cases in 2010/11 was 35 years, compared with 20 years during the pandemic (p = <0.0001). Conclusions/Significance The Health Protection Agency successfully established a sentinel surveillance system for severe influenza in 2010/11, detecting a rise in influenza cases mirroring other surveillance indicators. The data indicate an upward shift in the age-distribution of influenza A/H1N1 (2009) during the 2010/11 influenza season as compared to the 2009/10 pandemic. Systems to enable the ongoing surveillance of severe influenza will be a key component in understanding and responding to the evolving epidemiology of influenza in the post-pandemic era.

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

OBJECTIVES Ontario 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. METHODS A 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. RESULTS Eighteen 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. CONCLUSIONS This 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.

Antimalarial Therapy Selection for Quinolone Resistance among Escherichia coli in the Absence of Quinolone Exposure, in Tropical South America

Background Bacterial resistance to antibiotics is thought to develop only in the presence of antibiotic pressure. Here we show evidence to suggest that fluoroquinolone resistance in Escherichia coli has developed in the absence of fluoroquinolone use. Methods Over 4 years, outreach clinic attendees in one moderately remote and five very remote villages in rural Guyana were surveyed for the presence of rectal carriage of ciprofloxacin-resistant Gram-negative bacilli (GNB). Drinking water was tested for the presence of resistant GNB by culture, and the presence of antibacterial agents and chloroquine by HPLC. The development of ciprofloxacin resistance in E. coli was examined after serial exposure to chloroquine. Patient and laboratory isolates of E. coli resistant to ciprofloxacin were assessed by PCR-sequencing for quinolone-resistance-determining-region (QRDR) mutations. Results In the very remote villages, 4.8% of patients carried ciprofloxacin-resistant E. coli with QRDR mutations despite no local availability of quinolones. However, there had been extensive local use of chloroquine, with higher prevalence of resistance seen in the villages shortly after a Plasmodium vivax epidemic (p<0.01). Antibacterial agents were not found in the drinking water, but chloroquine was demonstrated to be present. Chloroquine was found to inhibit the growth of E. coli in vitro. Replica plating demonstrated that 2-step QRDR mutations could be induced in E. coli in response to chloroquine. Conclusions In these remote communities, the heavy use of chloroquine to treat malaria likely selected for ciprofloxacin resistance in E. coli. This may be an important public health problem in malarious areas.

Effectiveness of pertussis vaccination and duration of immunity

Background: A resurgence of pertussis cases among both vaccinated and unvaccinated people raises questions about vaccine effectiveness over time. Our objective was to study the effectiveness of the pertussis vaccine and characterize the effect of waning immunity and whole-cell vaccine priming. Methods: We used the test-negative design, a nested case–control study with test-negative individuals as controls. We constructed multivariable logistic regression models to estimate odds ratios (ORs). Vaccine effectiveness was calculated as (1 – OR) × 100. We assessed waning immunity by calculating the odds of developing pertussis per year since last vaccination and evaluated the relative effectiveness of priming with acellular versus whole-cell vaccine. Results: Between Dec. 7, 2009, and Mar. 31, 2013, data on 5867 individuals (486 test-positive cases and 5381 test-negative controls) were available for analysis. Adjusted vaccine effectiveness was 80% (95% confidence interval [CI] 71% to 86%) at 15–364 days, 84% (95% CI 77% to 89%) at 1–3 years, 62% (95% CI 42% to 75%) at 4–7 years and 41% (95% CI 0% to 66%) at 8 or more years since last vaccination. We observed waning immunity with the acellular vaccine, with an adjusted OR for pertussis infection of 1.27 (95% CI 1.20 to 1.34) per year since last vaccination. Acellular, versus whole-cell, vaccine priming was associated with an increased odds of pertussis (adjusted OR 2.15, 95% CI 1.30 to 3.57). Interpretation: We observed high early effectiveness of the pertussis vaccine that rapidly declined as time since last vaccination surpassed 4 years, particularly with acellular vaccine priming. Considering whole-cell vaccine priming and/or boosters in pregnancy to optimize pertussis control may be prudent.

What to do about pertussis vaccines? Linking what we know about pertussis vaccine effectiveness, immunology and disease transmission to create a better vaccine

Pertussis (whooping cough) is a respiratory disease caused by the bacterium Bordetella pertussis. Despite the implementation of immunization programs and high vaccine coverage in most jurisdictions, pertussis is still one of the most common vaccine-preventable diseases, suggesting that the current vaccines and immunization schedules have not been sufficiently effective. Several factors are thought to contribute to this. The acellular pertussis vaccine that has been used in many jurisdictions since the 1990s is less effective than the previously used whole-cell vaccine, with immunity waning over time. Both whole-cell and acellular pertussis vaccines are effective at reducing disease severity but not transmission, resulting in outbreaks in vaccinated cohorts. In this review, we discuss various limitations of the current approaches to protection from pertussis and outline various options for reducing the burden of pertussis on a population level.

Population-Level Impact of Ontario’s Infant Rotavirus Immunization Program: Evidence of Direct and Indirect Effects

Objective To evaluate the direct and indirect population impact of rotavirus (RV) immunization on hospitalizations and emergency department (ED) visits for acute gastroenteritis (AGE) in Ontario before and after the publicly-funded RV immunization program. Methods Administrative data was used to identify ED visits and hospitalizations for all Ontarians using ICD-10 codes. We used two outcome definitions: RV-specific AGE (RV-AGE) and codes representing RV-, other viral and cause unspecified AGE (“overall AGE”). The pre-program and public program periods were August 1, 2005 to July 31, 2011; and August 1, 2011 to March 31, 2013, respectively. A negative binominal regression model that included the effect of time was used to calculate rates and rate ratios (RRs) and 95% confidence intervals (CIs) for RV-AGE and overall AGE between periods, after adjusting for age, seasonality and secular trends. Analyses were conducted for all ages combined and age stratified. Results Relative to the pre-program period, the adjusted RRs for RV-AGE and overall AGE hospitalizations in the public program period were 0.29 (95%CI: 0.22–0.39) and 0.68 (95%CI: 0.62–0.75), respectively. Significant reductions in RV-AGE hospitalizations were noted overall and for the following age bands: < 12 months, 12–23 months, 24–35 months, 3–4 years, and 5–19 years. Significant declines in overall AGE hospitalizations were observed across all age bands, including older adults > = 65 years (RR 0.80, 95%CI: 0.72–0.90). The program was associated with adjusted RRs of 0.32 (95% CI: 0.20–0.52) for RV-AGE ED visits and 0.90 (95% CI: 0.85–0.96) for overall AGE ED visits. Conclusions This large, population-based study provides evidence of the impact of RV vaccine in preventing hospitalizations and ED visits for RV-AGE and overall AGE, including herd effects.

The basic reproduction number (R0) of measles: a systematic review

The basic reproduction number, R nought (R0), is defined as the average number of secondary cases of an infectious disease arising from a typical case in a totally susceptible population, and can be estimated in populations if pre-existing immunity can be accounted for in the calculation. R0 determines the herd immunity threshold and therefore the immunisation coverage required to achieve elimination of an infectious disease. As R0 increases, higher immunisation coverage is required to achieve herd immunity. In July, 2010, a panel of experts convened by WHO concluded that measles can and should be eradicated. Despite the existence of an effective vaccine, regions have had varying success in measles control, in part because measles is one of the most contagious infections. For measles, R0 is often cited to be 12-18, which means that each person with measles would, on average, infect 12-18 other people in a totally susceptible population. We did a systematic review to find studies reporting rigorous estimates and determinants of measles R0. Studies were included if they were a primary source of R0, addressed pre-existing immunity, and accounted for pre-existing immunity in their calculation of R0. A search of key databases was done in January, 2015, and repeated in November, 2016, and yielded 10 883 unique citations. After screening for relevancy and quality, 18 studies met inclusion criteria, providing 58 R0 estimates. We calculated median measles R0 values stratified by key covariates. We found that R0 estimates vary more than the often cited range of 12-18. Our results highlight the importance of countries calculating R0 using locally derived data or, if this is not possible, using parameter estimates from similar settings. Additional data and agreed review methods are needed to strengthen the evidence base for measles elimination modelling.

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.

Correlation of Real Time PCR Cycle Threshold Cut-Off with Bordetella pertussis Clinical Severity.

Bordetella pertussis testing performed using real-time polymerase chain reaction (RT-PCR) is interpreted based on a cycle threshold (Ct) value. At Public Health Ontario Laboratories (PHOL), a Ct value <36 is reported as positive, and Ct values ≥36 and <40 are reported as indeterminate. PHOL reported indeterminate results to physicians and public health units until May 2012, after which these results were only reported to physicians. We investigated the association between Ct value and disease symptom and severity to examine the significance of indeterminate results clinically, epidemiologically and for public health reporting. B. pertussis positive and indeterminate RT-PCR results were linked to pertussis cases reported in the provincial Integrated Public Health Information System (iPHIS), using deterministic linkage. Patients with positive RT-PCR results had a lower median age of 10.8 years compared to 12.0 years for patients with indeterminate results (p = 0.24). Hospitalized patients had significantly lower Ct values than non-hospitalized patients (median Ct values of 20.7 vs. 31.6, p<0.001). The proportion of patients reporting the most indicative symptoms of pertussis did not differ between patients with positive vs. indeterminate RT-PCR results. Taking the most indicative symptoms of pertussis as the gold-standard, the positive predictive value of the RT-PCR test was 68.1%. RT-PCR test results should be interpreted in the context of the clinical symptoms, age, vaccination status, prevalence, and other factors. Further information on interpretation of indeterminate RT-PCR results may be needed, and the utility of reporting to public health practitioners should be re-evaluated.