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.

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.

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

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

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.

Influenza increases invasive meningococcal disease risk in temperate countries.

OBJECTIVES: Invasive meningococcal disease (IMD) is a severe bacterial infection that displays wintertime seasonality in temperate countries. Mechanisms driving seasonality are poorly understood and may include environmental conditions and/or respiratory virus infections. We evaluated the contribution of influenza and environmental conditions to IMD risk, using standardized methodology, across multiple geographical regions. METHODS: We evaluated 3276 IMD cases occurring between January 1999 and December 2011 in 11 jurisdictions in Australia, Canada, France and the United States. Effects of environmental exposures and normalized weekly influenza activity on IMD risk were evaluated using a case-crossover design. Meta-analytic methods were used to evaluate homogeneity of effects and to identify sources of between-region heterogeneity. RESULTS: After adjustment for environmental factors, elevated influenza activity at a 2-week lag was associated with increased IMD risk (adjusted odds ratio (OR) per standard deviation increase 1.29; 95% confidence interval, 1.04-1.59). This increase was homogeneous across the jurisdictions studied. By contrast, although associations between environmental exposures and IMD were identified in individual jurisdictions, none was generalizable. CONCLUSIONS: Using a self-matched design that adjusts for both coseasonality and case characteristics, we found that surges in influenza activity result in an acute increase in population-level IMD risk. This effect is seen across diverse geographic regions in North America, France and Australia. The impact of influenza infection on downstream meningococcal risk should be considered a potential benefit of influenza immunization programmes.

Detection of Neisseria gonorrhoeae and Chlamydia trachomatis in pharyngeal and rectal specimens using the BD Probetec ET system, the Gen-Probe Aptima Combo 2 assay and culture.

OBJECTIVES: This study compared the sensitivity and specificity of culture and two nucleic acid amplification tests (NAATs): the BD Probetec ET system (PT) and the Aptima Combo 2 (AC2) in detecting Neisseria gonorrhoeae (GC) and Chlamydia trachomatis (CT) in pharyngeal and rectal specimens. METHODS: Male subjects were prospectively recruited at an MSM clinic in Toronto, Canada. Pharyngeal and rectal specimens were obtained for GC and CT culture, PT and AC2. Urine was also obtained for PT. A true positive was defined as: (1) positive culture, (2) positive PT and AC2 at the same site or (3) a single positive NAAT and detection of the same organism by any method at another site. RESULTS: 248 subjects were recruited. The prevalence of pharyngeal GC was 8.1%, rectal GC 11.7%, pharyngeal CT 2.0% and rectal CT 7.7%. The sensitivity of culture for pharyngeal GC and CT was 0%; 41.4% for rectal GC and 21.1% for rectal CT. The sensitivity of PT for pharyngeal GC, rectal GC, pharyngeal CT and rectal CT was 95.0%, 93.1%, 80.0% and 94.7%, respectively. The sensitivity of AC2 was 95.0% for pharyngeal GC and 100% at all other sites. Specificity was consistently above 98%. CONCLUSIONS: PT and AC2 detected GC and CT with superior sensitivity compared to culture. They detected 73 pharyngeal or rectal GC and CT infections compared to 16 by culture, using a rigorous gold standard. NAATs should be the method of choice for the detection of GC and CT in extragenital sites in men who have sex with men.

Use of Bordetella pertussis BP3385 to establish a cutoff value for an IS481-targeted real-time PCR assay.

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

Interim laboratory testing guidance for the detection of non-tuberculous Mycobacterium (NTM) infections in post-operative patients exposed to heater-cooler units.

The advice contained in this document should be read in conjunction with relevant federal, provincial, territorial and local legislation, regulations, and policies. Recommended measures should not be regarded as rigid standards, but principles and recommendations to inform the development of guidance. This advice is based on currently available scientific evidence and adopts a precautionary approach where the evidence is lacking or inconclusive. It was approved for publication on December 5, 2016. It is subject to review and change as new information becomes available. The main changes to this version include additions to: Case load reported to date, Sarcoidosis-like disease as an Indicator, Whole Genome Sequencing effort, links to Provincial and Territorial Lab Services and Health Canada reporting.

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.

Molecular phylogeny and evolutionary timescale for the family of mammalian herpesviruses.

A detailed phylogenetic analysis for mammalian members of the family Herpesviridae, based on molecular sequences is reported. Sets of encoded amino acid sequences were collected for eight well conserved genes that are common to mammalian herpesviruses. Phylogenetic trees were inferred from alignments of these sequence sets using both maximum parsimony and distance methods, and evaluated by bootstrap analysis. In all cases the three recognised subfamilies (Alpha-, Beta- and Gammaherpesvirinae), and major sublineages in each subfamily, were clearly distinguished, but within sublineages some finer details of branching were incompletely resolved. Multiple-gene sets were assembled to give a broadly based tree. The root position of the tree was estimated by assuming a constant molecular clock and also by analysis of one herpesviral gene set (that encoding uracil-DNA glycosylase) using cellular homologues as outgroups. Both procedures placed the root between the Alphaherpesvirinae and the other two subfamilies. Substitution rates were calculated for the combined gene sets based on a previous estimate for alphaherpesviral UL27 genes, where the time base had been obtained according to the hypothesis of cospeciation of virus and host lineages. Assuming a constant molecular clock, it was then estimated that the three subfamilies arose approximately 180 to 220 million years ago, that major sublineages within subfamilies were probably generated before the mammalian radiation of 80 to 60 million years ago, and that speciations within sublineages took place in the last 80 million years, probably with a major component of cospeciation with host lineages.

An international outbreak of salmonellosis associated with raw almonds contaminated with a rare phage type of Salmonella enteritidis.

During the winter of 2000 to 2001, an outbreak due to Salmonella Enteritidis (SE) phage type 30 (PT30), a rare strain, was detected in Canada. The ensuing investigation involved Canadian and American public health and food regulatory agencies and an academic research laboratory. Enhanced laboratory surveillance, including phage typing and pulsed-field gel electrophoresis, was used to identify cases. Case questionnaires were administered to collect information about food and environmental exposures. A case-control study with 16 matched case-control pairs was conducted to test the hypothesis of an association between raw whole almond consumption and infection. Almond samples were collected from case homes, retail outlets, and the implicated processor, and environmental samples were collected from processing equipment and associated farms for microbiological testing. One hundred sixty-eight laboratory-confirmed cases of SE PT30 infection (157 in Canada, 11 in the United States) were identified between October 2000 and July 2001. The case-control study identified raw whole almonds as the source of infection (odds ration, 21.1; 95% confidence interval, 3.6 to infinity). SE PT30 was detected in raw whole natural almonds collected from home, retail, distribution, and warehouse sources and from environmental swabs of processing equipment and associated farmers' orchards. The frequent and prolonged recovery of this specific organism from a large agricultural area was an unexpected finding and may indicate significant diffuse contamination on these farms. Identification of almonds as the source of a foodborne outbreak is a previously undocumented finding, leading to a North American recall of this product and a review of current industry practices.

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.

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.

Methicillin-resistant Staphylococcus aureus carriage in a child care center following a case of disease. Toronto Child Care Center Study Group.

OBJECTIVES: To study the prevalence of methicillin sodium-resistant and methicillin-sensitive Staphylococcus aureus colonization in a child care center following the diagnosis of community-acquired methicillin-resistant S. aureus (MRSA) disease in a previously well 2 1/2-year-old attendee and to determine the optimal site of detection of S. aureus. DESIGN: Point prevalence survey and questionnaire administration. SETTING: A Toronto, Ontario, child care center. INTERVENTIONS: Parents were provided with general information. Consenting parents completed a questionnaire and permitted screening of their child at 1 or more of throat, nose, and perianal sites. Families of children who were culture positive for MRSA were offered screening and suppressive therapy. Nasal and perianal swabs were obtained from child care center staff and screened. RESULTS: Of 201 children, 164 (81.6%) had completed questionnaires and had undergone screening at 1 or more sites; 38 staff members (100%) completed questionnaires and were screened. A 26-month-old classroom contact with chronic dermatitis had MRSA detected only on perianal swab. Of 3 adult household contacts of the index case and 2 adult and 1 child contacts of the classroom contact, only the 7-year-old sibling of the classroom contact was positive for MRSA. By pulse-field gel electrophoresis, these isolates were identical and not related to any of the common strains circulating in regional health care institutions. Of 40 children with S. aureus (24.4%), 33 had cultures at 3 sites, of which the throat was more sensitive (22 [67%]) than the nostrils (15 [46%]) or perianal sites (8 [24%]). There was a tendency for higher carriage of S. aureus in children with certain risk factors, including personal hospitalization (prevalence ratio, 2.9; 95% confidence interval, 0.6-12.1), family member hospitalization (prevalence ratio, 2.0; 95% confidence interval, 0.6-6.6), and visiting the hospital emergency department (prevalence ratio, 3.2; 95% confidence interval, 0.7-14.5), all in the previous 6 months. CONCLUSIONS: To our knowledge, this is one of the first recognized cases of MRSA disease and apparent transmission in a child care center. Throat and perianal site screenings have a higher sensitivity in identifying children colonized with S. aureus than nasal culturing. Infection with MRSA should be suspected in disease unresponsive to standard antibiotic therapy.

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.

A prolonged outbreak of exfoliative toxin A-producing Staphylococcus aureus in a newborn nursery.

An outbreak of erythromycin-resistant, exfoliative toxin-producing Staphylococcus aureus infection in a neonatal unit is described. The organism was coagulase positive but staphyloslide negative, and this unusual phenotype facilitated early recognition of the organism in the routine laboratory. In the initial outbreak there were 77 probable or confirmed cases, with a peak attack rate of 66%. Increased infection control measures were put in place and attempts were made to identify a staff carrier. No carriers were found and the major outbreak subsided. Sporadic cases occurred over the following 10 months, until May 1992, when a colonized staffperson was discovered. She was treated and no further cases occurred. The causative organism was subjected to typing by phage, enterobacterial repetitive intergenic consensus sequence polymerase chain reaction, and pulsed-field gel electrophoresis with two separate enzymes. The phage typing and genomic tests confirmed the presence of the same clone in the unit for 9 months. The organism possessed genes encoding exfoliative toxin A as determined by polymerase chain reaction.

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.