Increase in ST-11 serogroup W Neisseria meningitidis invasive meningococcal disease in Canada, 2016-2018.

BACKGROUND: Many countries have experienced increases in invasive meningococcal disease (IMD) due to a serogroup W Neisseria meningitidis (MenW) strain of the multilocus sequence type (ST)-11 clonal complex (CC). MenW ST-11 was first reported in Ontario, Canada, in 2014. By 2016, this strain caused IMD in five provinces and was responsible for 18.8% of the IMD cases in Canada. OBJECTIVE: To provide an update on invasive MenW disease in Canada including the strain characteristics, specimen source of isolates, age, sex and geographic distribution of cases. METHODS: N. meningitidis from culture-positive IMD cases are routinely submitted to the National Microbiology Laboratory (NML) for serogroup, serotype, serosubtype and sequence type analysis. The data from January 1, 2016 to December 31, 2018 were analyzed by calculating the proportion of IMD cases caused by MenW compared with other serogroups. In addition, trends based on age, sex and geographic distribution of cases and specimen source of isolates were analyzed based on information on specimen requisition forms. RESULTS: Over the 3-year period, 292 individual IMD case isolates were analyzed. The percentage of IMD case isolates typed as MenW more than doubled from 19% (n=15) to 44% (n=51) in 2018 when MenW became the most common serogroup, exceeded the number of MenB, MenC or MenY. In total, 93 MenW case isolates were identified, 91% (n=85) belonged to the ST-11 CC. The increase in MenW affected all age groups (but was most common in those older than 60) and both sexes, and occurred across the country but most prevalent in western Canada. The most common specimen source was blood. CONCLUSION: In 2018, MenW was the most common serogroup for isolates received by the NML from culture-positive IMD cases in Canada. Over 90% of the MenW serogroup isolates belonged to the ST-11 CC. The quadrivalent ACWY meningococcal conjugate vaccine protects against IMD caused by strains in the A, C, W or Y serogroups and therefore may protect against IMD caused by the new MenW ST-11 strain; however, more research is needed. The emergence of variant strains highlight the importance of strain characterization in IMD surveillance and research.

Multidrug-resistant and extensively drug-resistant Neisseria gonorrhoeae in Canada, 2012-2016.

BACKGROUND: Neisseria gonorrhoeae have acquired resistance to many antimicrobials, including third generation cephalosporins and azithromycin, which are the current gonococcal combination therapy recommended by the Canadian Guidelines on Sexually Transmitted Infections. OBJECTIVE: To describe antimicrobial susceptibilities for N. gonorrhoeae circulating in Canada between 2012 and 2016. METHODS: Antimicrobial resistance profiles were determined using agar dilution of N. gonorrhoeae isolated in Canada 2012-2016 (n=10,167) following Clinical Laboratory Standards Institute guidelines. Data were analyzed by applying multidrug-resistant gonococci (MDR-GC) and extensively drug-resistant gonococci (XDR-GC) definitions. RESULTS: Between 2012 and 2016, the proportion of MDR-GC increased from 6.2% to 8.9% and a total of 19 cases of XDR-GC were identified in Canada (0.1%, 19/18,768). The proportion of isolates with decreased susceptibility to cephalosporins declined between 2012 and 2016 from 5.9% to 2.0% while azithromycin resistance increased from 0.8% to 7.2% in the same period. CONCLUSION: While XDR-GC are currently rare in Canada, MDR-GC have increased over the last five years. Azithromycin resistance in N. gonorrhoeae is established and spreading in Canada, exceeding the 5% level at which the World Health Organization states an antimicrobial should be reviewed as an appropriate treatment. Continued surveillance of antimicrobial susceptibilities of N. gonorrhoeae is necessary to inform treatment guidelines and mitigate the impact of resistant gonorrhea.

Canadian recommendations for laboratory interpretation of multiple or extensive drug resistance in clinical isolates of Enterobacteriaceae, Acinetobacter species and Pseudomonas aeruginosa.

The goal of this document was to provide Canadian laboratories with a framework for consistent reporting and monitoring of multidrug resistant organisms (MDRO) and extensively drug resistant organisms (XDRO) for common gram-negative pathogens. This is the final edition of the interim recommendations, which were modified after one year of broad consultative review. This edition represents a consensus of peer-reviewed information and was co-authored by the Canadian Public Health Laboratory Network and the Canadian Association of Clinical Microbiology and Infectious Diseases. There are two main recommendations. The first recommendation provides standardized definitions for MDRO and XDRO for gram-negative organisms in clinical specimens. These definitions were limited to antibiotics that are commonly tested clinically and, to reduce ambiguity, resistance (rather than non-susceptibility) was used to calculate drug resistance status. The second recommendation identifies the use of standardized laboratory reporting of organisms identified as MDRO or XDRO. Through the broad consultation, which included public health and infection prevention and control colleagues, these definitions are ready to be applied for policy development. Both authoring organizations intend to review these recommendations regularly as antibiotic resistance testing evolves in Canada.

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.

Canadian Public Health Laboratory Network position statement: Non-culture based diagnostics for gastroenteritis and implications for public health investigations.

As clinical laboratories transition to using culture-independent detection test (CIDT) panels for cases of acute gastroenteritis, culture of clinical specimens is becoming less common. The reduction in bacterial cultures available for public health activities is expected to hinder surveillance and outbreak response by public health laboratories at the local, provincial, national and international levels. These recommendations are intended to serve as guidelines for the implementation of CIDT panels in frontline laboratories in Canada. The United States of America has already seen a significant reduction in culture of stool specimens despite the Association of Public Health Laboratories recommendation to perform reflex culture on positive CIDT specimens. Priority public health organisms addressed in these Canadian guidelines include Shiga toxin-producing Escherichia coli, Shigella and Salmonella and, under regional circumstances, other organisms such as Campylobacter jejuni/coli and Yersinia enterocolitica. These recommendations suggest active engagement between primary diagnostic laboratories and provincial public health laboratories to determine the workflow and protocols for reflex or parallel culture. Consequently, notifiable disease definitions will also need modification, with consultation of all stakeholders. Stakeholders need to work together to enhance recovery of bacterial isolates with best practices used for stool transport and storage.

Comparative genomic fingerprinting of Campylobacter: application in routine public health surveillance and epidemiological investigations.

A subtyping methodology for Campylobacter, Comparative Genomic Fingerprinting (CGF40), has been described recently. The objective of this study was to assess the utility of CGF40 as a tool to enhance routine public health surveillance of campylobacteriosis. Isolates of Campylobacter from across the province were requested and sent for CGF40 subtyping. Epidemiological data from cases reported to public health officials in Nova Scotia, Canada, from January 2012 to March 2015 were linked with blinded CGF40 subtyping results. CGF40 was epidemiologically valid; subtyping discerned known epidemiologically related isolates and augmented case-finding. Predominant sources and locations of subtype detection from the national reference database showed some study subtypes were rare and even novel to the database, while others were more commonly identified over multiple years and with exposures locally and internationally. A case-case study design was applied to examine risk factors for the most common CGF40 subtypes detected. Differences in the epidemiology of different CGF40 subtypes were observed. Statistically significant associations were noted for specific subtypes with rural residence, local exposure, contact with a pet dog or cat, contact with chickens, and drinking unpasteurized milk. With prospective use, CGF40 could potentially identify unrecognized outbreaks and contribute to epidemiological investigations of case clusters.

Decline in Decreased Cephalosporin Susceptibility and Increase in Azithromycin Resistance in Neisseria gonorrhoeae, Canada.

Antimicrobial resistance profiles were determined for Neisseria gonorrhoeae strains isolated in Canada during 2010-2014. The proportion of isolates with decreased susceptibility to cephalosporins declined significantly between 2011 and 2014, whereas azithromycin resistance increased significantly during that period. Continued surveillance of antimicrobial drug susceptibilities is imperative to inform treatment guidelines.

Molecular Assay for Detection of Ciprofloxacin Resistance in Neisseria gonorrhoeae Isolates from Cultures and Clinical Nucleic Acid Amplification Test Specimens.

We developed a real-time PCR assay to detect single nucleotide polymorphisms associated with ciprofloxacin resistance in specimens submitted for nucleic acid amplification testing (NAAT). All three single nucleotide polymorphism (SNP) targets produced high sensitivity and specificity values. The presence of ≥2 SNPs was sufficient to predict ciprofloxacin resistance in an organism.

Molecular Assay for Detection of Genetic Markers Associated with Decreased Susceptibility to Cephalosporins in Neisseria gonorrhoeae.

The incidence of antimicrobial-resistant Neisseria gonorrhoeae continues to rise in Canada; however, antimicrobial resistance data are lacking for approximately 70% of gonorrhea infections that are diagnosed directly from clinical specimens by nucleic acid amplification tests (NAATs). We developed a molecular assay for surveillance use to detect mutations in genes associated with decreased susceptibility to cephalosporins that can be applied to both culture isolates and clinical samples. Real-time PCR assays were developed to detect single nucleotide polymorphisms (SNPs) in ponA, mtrR, penA, porB, and one N. gonorrhoeae-specific marker (porA). We tested the real-time PCR assay with 252 gonococcal isolates, 50 nongonococcal isolates, 24 N. gonorrhoeae-negative NAAT specimens, and 34 N. gonorrhoeae-positive NAAT specimens. Twenty-four of the N. gonorrhoeae-positive NAAT specimens had matched culture isolates. Assay results were confirmed by comparison with whole-genome sequencing data. For 252 N. gonorrhoeae strains, the agreement between the DNA sequence and real-time PCR was 100% for porA, ponA, and penA, 99.6% for mtrR, and 95.2% for porB. The presence of ≥2 SNPs correlated with decreased susceptibility to ceftriaxone (sensitivities of >98%) and cefixime (sensitivities of >96%). Of 24 NAAT specimens with matched cultures, the agreement between the DNA sequence and real-time PCR was 100% for porB, 95.8% for ponA and mtrR, and 91.7% for penA. We demonstrated the utility of a real-time PCR assay for sensitive detection of known markers for the decreased susceptibility to cephalosporins in N. gonorrhoeae. Preliminary results with clinical NAAT specimens were also promising, as they correlated well with bacterial culture results.

Pyrosequencing assay to rapidly detect clarithromycin resistance mutations in Canadian Helicobacter pylori isolates.

BACKGROUND: Mutations at positions 2142 or 2143 in the twocopy 23S ribosomal RNA gene of Helicobacter pylori are highly predictive of in vitro clarithromycin resistance and failure of clarithromycin-containing treatment regimens. OBJECTIVE: To design an assay to rapidly detect these mutations using rapid polymerase chain reaction and pyrosequencing, a novel method of 'sequencing by synthesis', and to test this assay with a collection of Canadian H pylori isolates. METHODS: Forty-two H pylori isolates (24 clarithromycin-resistant, 18 clarithromycin-susceptible) were studied. A target region in the 23S gene was rapidly amplified and sequenced by pyrosequencing. RESULTS: Mutations at one of the two positions studied were present in 20 of the 24 (83%) clarithromycin-resistant isolates; 13 had double- copy A2143G mutations, four had double-copy A2142G mutations and three had single-copy A2143G mutations. There were no mutations in 17 of the 18 (94%) susceptible isolates. A single-copy A2142G mutation was detected in one susceptible isolate. CONCLUSIONS: The pyrosequencing assay developed was able to detect and differentiate mutations at positions 2142 and 2143 in either one or both copies of the H pylori 23S ribosdomal RNA gene. Further study is needed to determine whether this pyrosequencing assay can be used to determine H pylori susceptibility to clarithromycin from clinical specimens such as stools or gastric biopsies.

Multilaboratory comparison of proficiencies in susceptibility testing of Helicobacter pylori and correlation between agar dilution and E test methods.

Susceptibility testing was performed at seven Canadian microbiology laboratories and the Helicobacter Reference Laboratory, Halifax, Nova Scotia, Canada, to assess susceptibility testing proficiency and the reproducibility of the results for clarithromycin and metronidazole and to compare the Epsilometer test (E test) method to the agar dilution reference method. Control strain Helicobacter pylori ATCC 43504 (American Type Culture Collection) and 13 clinical isolates (plus duplicates of four of these strains including ATCC 43504) were tested blindly. The National Committee for Clinical Laboratory Standards (NCCLS) guidelines for agar dilution testing were followed, and the same suspension of organisms was used for agar dilution and E test. Antimicrobials and E test strips were provided to the investigators. Methods were provided on a website (www.Helicobactercanada.org). Each center reported MICs within the stated range for strain ATCC 43504. Compared to the average MICs, interlaboratory agreements within 2 log(2) dilutions were 90% (range, 69 to 100%) for clarithromycin by agar dilution, with seven very major errors [VMEs], and 85% (range, 65 to 100%) by E test, with three VMEs. Interlaboratory agreements within 2 log(2) dilutions were 83% (range, 50 to 100%) for metronidazole by agar dilution, with six VMEs and eight major errors (MEs), and 75% (range, 50 to 94%) by E test, with four VMEs and four MEs. At lower and higher concentrations of antibiotic, E test MICs were slightly different from agar dilution MICs, but these differences did not result in errors. When a standardized protocol based on NCCLS guidelines was used, most participants in this study correctly identified clarithromycin- and metronidazole-susceptible and -resistant strains of H. pylori 93% of the time by either the agar dilution or E test method, and the numbers of errors were relatively equivalent by both methods.

Comparison of the Velogene Rapid MRSA Identification Assay, Denka MRSA-Screen Assay, and BBL Crystal MRSA ID System for rapid identification of methicillin-resistant Staphylococcus aureus.

Early detection of methicillin-resistant S.aureus (MRSA) is critical for both the management of infected patients, and the timely institution of appropriate infection control measures. Although detection of the mecA gene by PCR remains the gold standard, this technology is inaccessible for many laboratories. Therefore, we sought to evaluate several new rapid identification systems and compare them to PCR. A total of 71 methicillin-susceptible S. aureus (MSSA), 25 borderline oxacillin-resistant S. aureus (BORSA), and 213 MRSA were selected for study. S.aureus was identified using standard methods. Initial screening was performed on a Mueller-Hinton agar plate with 6 mg/L of oxacillin. MRSA strains were identified using PCR with primers specific for the mecA gene. PCR was used as the reference method. All isolates were tested using the BBL Crystal MRSA ID System (Becton Dickinson Microbiology Systems, Maryland, USA), the MRSA-Screen Assay (Denka Seiken Co., Ltd., Tokyo, Japan), and the Velogene Rapid MRSA Identification Assay (ID Biomedical Corp, Vancouver, BC). With minor modifications, all assays were performed according to manufacturers' instructions. Overall, the 3 commercial assays performed well. The sensitivity and specificity of the BBL, Denka, and Velogene systems were 99%/100%, 99%/100%, and 96%/100% respectively. The advantages of the phenotypic tests-BBL Crystal Kit and Denka MRSA-Screen Assay include lower cost per test, shelf-life, ease of use, and rapid turn-around times. Advantages of the Velogene Rapid MRSA include ability to perform genotypic high-volume testing without the equipment requirements and technical complexity involved with PCR. Turn-around times ranged from 15 min for the Denka MRSA-Screen Assay, 2 h for the Velogene Rapid MRSA, and 4 h for the BBL Crystal. The BBL Crystal, Denka MRSA-Screen, and Velogene Rapid MRSA identification systems are rapid, easy to perform, and provide accurate identification of MRSA. These rapid kits offer an acceptable alternative for smaller, non-reference, laboratories and reduce the dependency on PCR in larger laboratories for routine confirmation.

Four-day incubation for detection of bacteremia using the BACTEC 9240.

Records of 29,356 blood cultures performed between April 1994 and April 1997, using the BACTEC 9240 continuous monitoring blood culture system, were reviewed retrospectively. From these, 3,127 blood culture vials became positive. Of 95 blood culture isolates detected after three days of incubation, 63 were recovered on day four and 32 on day five. Twenty-six contaminants were recovered on day four, and 21 on day five. Chart review was performed for all day four and five isolates that did not meet our definition of a contaminant. Of the 40 isolates that were clinically insignificant, 31 were recovered on day four, and nine on day five. Of eight clinically significant isolates, six were recovered on day four, and two on day five. Our data support a four-day incubation protocol for the recovery of all clinically significant bacteria with overall sensitivity reduced by only 0.06% when compared with a five-day protocol.

Cross-Canada spread of methicillin-resistant Staphylococcus aureus via transplant organs.

We report our investigation of the transmission of methicillin-resistant Staphylococcus aureus (MRSA) through transplantation. The kidneys, liver, and corneas were harvested from a child who died in Nova Scotia. Several days postmortem it was learned that culture of a premortem endotracheal tube aspirate from the donor yielded MRSA. Both kidneys were transplanted into a child in Nova Scotia and the liver into a child in Alberta. Both recipients subsequently became blood culture-positive for MRSA. One corneal ring from the donor was MRSA-positive. All four MRSA isolates were mecA-positive by polymerase chain reaction (PCR). The relatedness of the MRSA isolates was examined by restriction fragment length polymorphism (RFLP) analysis, a 16S-23S ribosomal PCR typing method, and comparison of antibiograms. Results were identical for all four MRSA isolates. These findings indicate that MRSA from the donor was transferred to recipients during implantation of harvested organs in Alberta and Nova Scotia, a cross-Canada spread.

A Canadian multicentre case-control study of sporadic Escherichia coli 0157:H7 infection.

OBJECTIVE: To evaluate further risk factors for Escherichia coli 0157:H7 infection including consumer preferences related to the consumption of ground beef and the role of person-to-person transmission of this infection. PATIENTS AND METHODS: A case-control study of sporadic E coli 0157:H7 infection was undertaken in five Canadian cites from June to December 1991. One hundred cases of E coli 0157:H7 infection were age- and sex-matched to 200 neighbourhood controls. Cases and controls were interviewed face-to-face to obtain information on potential risk factors for infection and health outcomes. Daycare providers of case and control children were interviewed regarding risk factors for infection at the institutional level. Contacts of cases and controls who reported diarrhea in the seven days before the case onset date were also interviewed about their symptoms and risk factors. RESULTS: All cases had diarrhea during the course of their illness and 90 (90%) reported bloody diarrhea. Four (4%) were reported to have developed hemolytic uremic syndrome; however, there were no fatalities. Sixty-one (61%) of patients were hospitalized. Two variables were associated with infection in the final conditional logistic regression model: eating pink hamburger patties (odds ratio = 12.4, P=0.0001, population attributable fraction =40.2%) and contact with a nonhousehold member suffering from diarrhea (odds ratio = 7.0, P=0.0054, population attributable fraction = 10.3%) in the seven days before illness. Forty per cent of cases and controls who indicated that they prefer well done hamburgers said they would eat a 'pink' hamburger if served to them rather than ask that the hamburger be cooked longer. CONCLUSIONS: Health care workers should remain vigilant in their efforts to educate the public as to the risks associated with the consumption of ground beef that is inadequately cooked, and the importance of personal hygiene in the prevention of enteric illness.

Helicobacter pylori: primary susceptibility to clarithromycin in vitro in Nova Scotia.

Resistance to antimicrobial agents is a major determinant of the efficacy of regimens to eradicate Helicobacter pylori. Clarithromycin (CLA) has become one of the most commonly used antibiotics for treatment of H pylori infection. In this study, the rate of primary resistance to CLA in H pylori isolated from patients was determined. One hundred sixty-two strains were recovered from patients before treatment. Strains were grown and inoculated onto Mueller-Hinton agar with 7% sheep blood. CLA epsilometer gradient agar diffusion test (E test) strips were used to test for susceptibility. Appropriate control organisms were tested to validate the assay. Plates were incubated at 37 degrees C in a microaerophilic atmosphere for up to five days. E test results were easy to interpret. Strains were considered resistant if the minimum inhibitory concentration (MIC) was 2 micrograms/mL or greater. Three strains were resistant (two strains with MIC 8 micrograms/mL and one strain with MIC 12 micrograms/mL) and 159 strains were sensitive (MICs ranged from less than 0.016 to 0.38 micrograms/mL). Ninety per cent of the strains had MICs of 0.023 micrograms/mL. Primary resistance was 1.8%. These susceptibility data support the use of CLA for the treatment of H pylori in the Nova Scotia population.

Subcutaneous dirofilariasis in Nova Scotia.

A case of dirofilariasis in a 78-year-old woman from Nova Scotia is described along with the histological findings and the basis of identification of the parasite. The patient developed a subcutaneous nodule, which was excised. Dirofilaria ursi and ursi-like nematodes are a rare cause of subcutaneous nodules. This is the first time dirofilariasis has been diagnosed in an adult in Atlantic Canada.