Bacillus Calmette-Guérin strains with defined resistance mutations: a new tool for tuberculosis laboratory quality control.

OBJECTIVE: Laboratory quality control (QC) is essential to assess the reliability of tuberculosis diagnostic testing. To provide safe QC reagents for the detection of drug-resistant Mycobacterium tuberculosis, we generated antibiotic-resistant mycobacterial strains of attenuated virulence (M. bovis bacillus Calmette-Guérin (BCG)). METHODS: Seven mono-resistant BCG strains were developed by introducing resistance-conferring mutations into wild-type BCG strains. Mutations were confirmed by dideoxynucleotide sequencing. Phenotypic resistance was quantified by microbroth dilution to determine the MIC90. The capacity of two commercial tests (GeneXpert TB/RIF and Genotype MTBDRplus) to detect resistance-conferring mutations was evaluated independently. RESULTS: Our panel included BCG strains with mutations in rpoB (S450L, I491F), katG (deletion at AA428), gyrA (D94G), rpsL (K43R) and Rv0678c (S63R). These mutations translated respectively into phenotypic resistance to rifampin (MIC ≥8 mg/L), isoniazid (MIC ≥8 mg/L), moxifloxacin (MIC 4 mg/L) and streptomycin (MIC ≥8 mg/L); the Rv0678c mutant showed decreased susceptibility to both clofazimine (MIC 4 mg/L) and bedaqualine (MIC 1 mg/L). GeneXpert (Cepheid) and Genotype MTBDRplus (Hain Lifesciences) both called the rpoB S450L strain rifampin-resistant and the I491F mutant rifampin-susceptible, as expected based on single nucleotide polymorphism positions. Likewise, MTBDRplus called the novel katG deletion mutant isoniazid susceptible despite phenotypic resistance. CONCLUSION: BCG strains engineered to be mono-resistant to anti-tuberculosis drugs can be used as safe QC reagents for tuberculosis diagnostics and drug susceptibility testing.

Invasive serogroup W Neisseria meningitidis (MenW) in Ontario, Canada shows potential clonal replacement during the period January 1, 2009 to June 30, 2016.

BACKGROUND: In Ontario, serogroup W Neisseria meningitidis (MenW) accounts for a small percentage of all invasive meningococcal disease (IMD) and between 2010 and 2014, only zero to three confirmed cases occurred per year. However, between August 2015 and June 2016, six culture confirmed MenW IMD cases were reported in Ontario. OBJECTIVE: All MenW IMD cases in Ontario between January 1, 2009 and June 30, 2016 were reviewed and the N. meningitidis strains involved were characterized. METHODS: MenW cases were identified in the Integrated Public Health Information System byf Public Health Ontario. MenW isolates were characterized at the National Microbiology Laboratory. RESULTS: Of the thirteen MenW IMD cases, six were due to isolates typed as sequence type (ST)-22 clonal complex (cc), six were of ST-11 cc, and one ST-167 cc. Most (83%) MenW cases due to the ST-22 cc occurred prior to 2012 while all six MenW cases due to ST-11 cc happened since May 2014. The six MenW ST-11 isolates appeared to be clonal. CONCLUSION: It appears that a genetic shift in the invasive MenW isolates has occurred in Ontario in 2014 with the ST-11 clone replacing the traditional ST-22 clone.

Epidemiology of serogroup C and Y invasive meningococcal disease (IMD) in Ontario, 2000-2013: Vaccine program impact assessment.

OBJECTIVES: A publicly-funded meningococcal serogroup C conjugate vaccine (MCCV) program was introduced in Ontario, in 2004/2005 for 1-year-old children as well as adolescents (approximately 12 years old). In 2009, quadrivalent meningococcal conjugate vaccine (MCV4) replaced MCCV for grade seven students. Our objective was to determine meningococcal vaccine program impact on reported cases of serogroup C and Y invasive meningococcal disease (IMD) at the population level in Ontario, Canada. METHODS: Data were obtained from the Ontario reportable diseases system, the integrated Public Health Information System (iPHIS), and Public Health Ontario Laboratories (PHOL) for 2000-2013. Descriptive epidemiologic analyses, including age-specific rates for age groups based on program eligibility, were conducted. Changes over the 14-year observation period and comparison of pre- and post-program periods for MCCV and MCV4 were assessed. Analyses were conducted using SAS 9.3. RESULTS: There were 161 serogroup C IMD cases and its annual incidence decreased significantly over time (17.2% reduction per year [95% CI: 13.4 to 20.7]). The incidence of serogroup C IMD decreased significantly in children aged 1-17 years in the post-program period, based on age-specific incidence rate ratios (IRRs) and their 95% confidence intervals (CIs). Adolescents 12-16 years had the lowest serogroup C IRR (0.07 [95% CI: 0.01 to 0.55]); the rate decreased more than 14-fold between the pre- and post-periods. There were 187 serogroup Y IMD cases and there was a non-significant 1.6% reduction per year [95% CI: -1.9 to 5.1]) over the surveillance period. Likewise, there was a non-significant decrease in serogroup Y IMD among persons 12-16 years (MCV4 eligible) in the post-program period. CONCLUSIONS: Reductions in serogroup C IMD among program eligible and ineligible age groups suggest both direct and indirect MCCV vaccine program impact. Continued surveillance of IMD in Ontario is important to further assess MCV4 program impact.

Whole-genome sequencing of the Mycobacterium tuberculosis Manila sublineage results in less clustering and better resolution than mycobacterial interspersed repetitive-unit-variable-number tandem-repeat (MIRU-VNTR) typing and spoligotyping.

Mycobacterium tuberculosis isolates of the Manila sublineage are genetically homogeneous. In this study, we used whole-genome sequencing (WGS) to type a collection of 36 M. tuberculosis isolates of the Manila family. WGS enabled the subtyping of these 36 isolates into at least 10 distinct clusters. Our results indicate that WGS is a powerful approach to determining the relatedness of Manila family M. tuberculosis isolates.

Profiling of rpoB mutations and MICs for rifampin and rifabutin in Mycobacterium tuberculosis.

Resistance to rifampin (RIF) and rifabutin (RFB) in Mycobacterium tuberculosis is associated with mutations within an 81-bp region of the rpoB gene (RIF resistance-determining region [RRDR]). Previous studies have shown that certain mutations in this region are more likely to confer high levels of RIF resistance, while others may be found in phenotypically susceptible isolates. In this study, we sought to determine the relationship between the MICs of RIF and RFB and rpoB RRDR mutations in 32 multidrug-resistant (MDR), 4 RIF-monoresistant, and 5 susceptible M. tuberculosis clinical isolates. The MICs were determined using the MGIT 960 system. Mutations in the rpoB RRDR were determined by Sanger sequencing. RpoB proteins with mutations S531L (a change of S to L at position 531), S531W, H526Y, and H526D and the double mutation D516A-R529Q were associated with high MICs for RIF and RFB. Five isolates carrying the mutations L511P, H526L, H526N, and D516G-S522L were found to be susceptible to RIF. Several mutations were associated with resistance to RIF and susceptibility to RFB (F514FF, D516V, and S522L). Whole-genome sequencing of two MDR isolates without rpoB RRDR mutations revealed a mutation outside the RRDR (V146F; RIF MIC of 50 µg/ml). The implications of the polymorphisms identified in the second of these isolates in RIF resistance need to be further explored. Our study further establishes a correlation between the mutations and the MICs of RIF and, also, RFB in M. tuberculosis. Several rpoB mutations were identified in RIF- and RFB-susceptible isolates. The clinical significance of these findings requires further exploration. Until then, a combination of phenotypic and molecular testing is advisable for drug susceptibility testing.

Epidemiological evaluation of spatiotemporal and genotypic clustering of Mycobacterium tuberculosis in Ontario, Canada.

BACKGROUND: In Canada, tuberculosis (TB) rates are at a historic low, with the remaining risk concentrated in a few vulnerable population subgroups. OBJECTIVES: To describe the epidemiology of TB in the Canadian province of Ontario and to characterise risk factors associated with transmission events, identified using genetic typing techniques. DESIGN: Retrospective analysis of 2186 culture-positive TB cases between August 2007 and December 2011. Temporal trends and risk of spatiotemporal and genotypic clustering were evaluated using Poisson and logistic regression models. RESULTS: Being in a spatiotemporal cluster was associated with Aboriginal status (odds ratio [OR] 3.63, 95% confidence interval [CI] 1.23-10.71). Cases in genotypic clusters were more likely to report homelessness as a risk factor (adjusted OR [aOR] 2.92, 95%CI 1.74-4.90) or be male (aOR 1.35, 95%CI 1.09-1.68), and were less likely to be aged ≥ 65 years (aOR 0.63, 95%CI 0.49-0.82), foreign-born (aOR 0.32, 95%CI 0.24-0.43) or Aboriginal (aOR 0.40, 95%CI 0.16-0.99). The Beijing lineage had an annual rate of increase of almost 10% (P = 0.047), and was associated with genotypic clustering (aOR 2.84, 95%CI 2.19-3.67). CONCLUSION: Genotypic data suggest that disease clusters are smaller, but far more common, than would be estimated using spatiotemporal clustering.

Burden of illness and factors associated with duration of illness in clinical campylobacteriosis.

A population-based study investigated the burden of illness, including the duration of illness associated with laboratory-confirmed cases of campylobacteriosis in two health unit areas. Questionnaire data were collected for 250 cases. The median duration of illness was 8 days and 66% of cases reported symptoms of moderate severity or greater. A Cox proportional hazards model identified antimicrobial use factors associated with a significantly increased rate of symptom resolution (shorter duration of illness): macrolides for less than the recommended number of days, ciprofloxacin for at least 3 days, and antimicrobials not recommended for campylobacteriosis. The impact of antimicrobial use was consistent regardless of when, during the course of illness, the antimicrobial use began. The effectiveness of ciprofloxacin in these results may be due to the low prevalence of resistance to ciprofloxacin in isolates from this study. The effect of antimicrobials not recommended for campylobacteriosis should be further investigated.

The role of drinking water in the transmission of antimicrobial-resistant E. coli.

To determine whether drinking water contaminated with antimicrobial-resistant E. coli is associated with the carriage of resistant E. coli, selected households sending water samples to Ontario and Alberta laboratories in 2005-2006 were asked to participate in a cross-sectional study. Household members aged ≥12 years were asked to complete a questionnaire and to submit a rectal swab. In 878 individuals, 41% carried a resistant strain of E. coli and 28% carried a multidrug-resistant strain. The risk of carriage of resistant E. coli was 1·26 times higher for users of water contaminated with resistant E. coli. Other risk factors included international travel [prevalence ratio (PR) 1·33], having a child in nappies (PR 1·33), being male (PR 1·33), and frequent handling of raw red meats (PR 1·10). Protecting private water sources (e.g. by improving systems to test and treat them) may help slow the emergence of antimicrobial resistance in E. coli.

Genotypic characterization of tuberculosis transmission within Toronto's under-housed population, 1997-2008.

Toronto has been the site of a recent extended tuberculosis (TB) outbreak in the homeless or under-housed population. Genotyping has identified a unique strain that continues to circulate within this population, with spread to individuals with no links to the shelter system, and anecdotally appears to progress rapidly from infection to active disease in some cases. The recent appearance and transmission of another unique strain was also identified, indicating that TB transmission continues to be a problem within the under-housed population. Enhanced surveillance utilizing molecular epidemiology is a useful tool to assist in TB control in vulnerable populations.

Changing epidemiology of invasive Haemophilus influenzae in Ontario, Canada: evidence for herd effects and strain replacement due to Hib vaccination.

The epidemiology of invasive Haemophilus influenzae infections was evaluated in Ontario between 1989 and 2007 to assess the impact of the introduction of the conjugate H. influenzae serotype b (Hib) vaccine in the early 1990 s on Hib and non-Hib serotypes in both vaccinated and unvaccinated cohorts as well as the possibility of "strain replacement" with non-vaccine H. influenzae strains. Data were collected by the provincial Public Health Laboratories-Toronto, Ontario Agency for Health Protection and Promotion, which performed almost all serotyping on invasive (blood, CSF, other sterile sites) H. influenzae strains isolated in the province during the study period. Temporal trends for Hib, other typeable strains, and non-typeable H. influenzae were evaluated by Poisson regression, controlling for the specimen submissions. Prior to infant Hib vaccination, the most commonly observed serotype was serotype b (64.9%). Subsequently, 70.3%, 13.6%, and 9.4% of isolates were non-typeable, serotype f, and serotype b, respectively. Infant Hib vaccination resulted in a decrease in Hib incidence in all age groups (pooled IRR 0.432) and marked increases of non-typeable and serotype f H. influenzae in children aged <5 years (IRR 2.4 and 3.0, respectively). Vaccination against Hib has altered the epidemiology of invasive H. influenzae infections in Ontario. Prevention of invasive Hib disease was observed in both vaccinated and unvaccinated age groups. Invasive H. influenzae infection now commonly presents as sepsis due to non-typeable H. influenzae in older individuals. However, strain replacement of Hib with serotype f and non-typeable strains in children under 5 years was documented.

Rapid molecular diagnosis of tuberculous meningitis using the Gen-probe Amplified Mycobacterium Tuberculosis direct test in a large Canadian public health laboratory.

OBJECTIVE: A 5-year retrospective study of the performance of the Gen-Probe Amplified Mycobacterium Tuberculosis Direct Test (MTD) for detecting Mycobacterium tuberculosis complex in cerebrospinal fluid (CSF). Patient data from culture-confirmed cases of tuberculous meningitis (TBM) were also analysed. RESULTS: In total, 311 CSF specimens were tested by the MTD, of which 17 were positive. When compared with culture (gold standard), the sensitivity and specificity of the MTD test were 93.8% and 99.3%, respectively. The positive and negative predictive values for TBM were 88.2%, and 99.7%. Clinical and epidemiological information was requested for all culture-positive TBM patients. These data were used to assess the mortality rate (55.6%) and to determine common factors that could be applied as selection criteria for the appropriate testing of CSF by MTD. CONCLUSION: The study found the MTD test to be a rapid, sensitive and specific test for TBM. A history of immigration from an area endemic for tuberculosis (TB), a history of TB, symptoms of neurological deficits and the results of CSF analyses could be used to appropriately select CSF for MTD testing in order to provide a critical early diagnosis of TBM.

Routine use of the Gen-Probe MTD2 amplification test for detection of Mycobacterium tuberculosis in clinical specimens in a large public health mycobacteriology laboratory.

The new version of the Amplified Mycobacterium Tuberculosis Direct Test, MTD2 (Gen-Probe Inc., San Diego, CA) has been implemented as part of the regular testing algorithm for detecting Mycobacterium tuberculosis (MTB) in selected respiratory and non-respiratory specimens in our laboratory. At the Central Public Health Laboratory, Etobicoke, Ontario, we receive specimens for the detection of mycobacteria from all areas of the Province of Ontario. The laboratory processes approximately 25,000 specimens per year, and receives approximately 2000 reference cultures for identification. There are 600 to 700 new cases of tuberculosis detected yearly. Over the 1-year period (1997-98), 823 specimens were tested by MTD2 and the results were compared with radiometric culture (Bactec, Becton Dickinson, Sparks, MD) and clinical diagnosis, giving an overall sensitivity, specificity, positive predictive value and negative predictive values of 100%, 99.6%, 97.4% and 100%, respectively. Two hundred and two cases of respiratory TB and 56 cases of extrapulmonary TB were detected by MTD2 within 0-4 days of specimen arrival in the laboratory. By appropriate selection of specimens for testing, the MTD2 can provide a fast, accurate, and cost-effective method for the detection of MTB in clinical specimens.

Human torovirus: a new nosocomial gastrointestinal pathogen.

Studies were undertaken to determine if human torovirus is associated with gastroenteritis and to examine the clinical features of torovirus illness in children. The fecal excretion of torovirus in patients with gastroenteritis was compared with that in matched asymptomatic controls in a case-control study. Toroviruses were identified in 72 (35.0%) of 206 gastroenteritis cases compared with 30 (14.5%) of 206 controls (P<.001). Clinical features of torovirus gastroenteritis in 172 patients positive for torovirus were compared with those of 115 patients infected with rotavirus or astrovirus. Persons infected with torovirus were more frequently immunocompromised (43.0% vs. 15.7%) and nosocomially infected (57.6% vs. 31.3%). They also experienced less vomiting (46.4% vs. 66.7%) but had more bloody diarrhea (11.2% vs. 1.8%). An antibody response to torovirus developed mainly in older, nonimmunocompromised children (P<.01). These studies demonstrate an association between torovirus excretion and gastroenteritis in the pediatric population among immunocompromised hospitalized patients and in previously healthy patients.

A cluster of surgical wound infections due to unrelated strains of group A streptococci.

Group A streptococci account for less than 1% of all surgical wound infections but are an important cause of nosocomial outbreaks. We report here a cluster of four group A streptococcal infections that occurred within an 11-day period on a single surgical service. The index case presented with toxic shock-like syndrome. Epidemiologic investigation did not identify any relationship between infections. Restriction endonuclease analysis and M and T typing found the four isolates to be unrelated. Restriction endonuclease analysis is a useful tool for determining relatedness of nosocomial isolates of group A streptococci.