Rapid Detection of KPC, NDM, and OXA-48-Like Carbapenemases by Real-Time PCR from Rectal Swab Surveillance Samples.

We describe a multiplex real-time PCR assay for use on the ABI 7500 Fast TaqMan platform to detect all currently described Klebsiella pneumoniae carbapenemases (KPC), New Delhi metallo-ß-lactamases (NDM), and the OXA-48-like family of carbapenemases from bacterial culture lysates or sample enrichment broth lysates.

Detection of enterovirus D68 in Canadian laboratories.

The recent emergence of a severe respiratory disease caused by enterovirus D68 prompted investigation into whether Canadian hospital and provincial laboratories can detect this virus using commercial and laboratory-developed assays. This study demonstrated analytical sensitivity differences between commercial and laboratory-developed assays for the detection of enterovirus D68.

An outbreak of norovirus caused by consumption of oysters from geographically dispersed harvest sites, British Columbia, Canada, 2004.

OBJECTIVES: In January 2004, an increase in gastrointestinal illness following oyster consumption was reported in British Columbia. An investigation was initiated to explore the association between norovirus infection and consumption of British Columbia oysters and to identify the source of oyster contamination. METHODS: The outbreak investigation included active surveillance for human cases, two cohort studies, trace-back of oysters, and laboratory testing of oysters and human stools. RESULTS: Enhanced surveillance identified 26 confirmed and 53 clinical cases over 3 months. Oyster consumption was associated with illness in one cohort and suggestive in the other. Oysters were traced to 14 geographically dispersed harvest sites, 18 suppliers, and 45 points of purchase. Norovirus BCCDC03-028 (genotype I.2) was detected in 50% of human specimens. Experimental methods detected norovirus in 12 oyster samples. Sequencing identified mixed clonal patterns in the oysters with one direct sequence match between an oyster sample and the associated human specimen. CONCLUSIONS: The consumption of raw oysters led to norovirus infection. The source of oyster contamination remained unidentified. The geographical dispersion of implicated harvest sites was unusual. APPLICATIONS: This outbreak is unlike most shellfish outbreaks that can be traced back to a common source and challenges conventional thinking that all oyster-related norovirus outbreaks of are a result of point source contamination.

Usefulness of pulsed-field gel electrophoresis in tracking two outbreaks of invasive meningococcal disease serogroup C in British Columbia.

Two major outbreaks of invasive meningococcal disease serogroup C (IMD-C) were identified in British Columbia between 2000 and 2004. Pulsed-field gel electrophoresis (PFGE) and porA gene sequencing of all retained IMD-C isolates were used to assess correlations between genotypes and epidemiological patterns. PFGE patterns of IMD-C genotypes correlated with epidemiological patterns between 2000 and 2004 in British Columbia, and demonstrated that PFGE can identify outbreak-related cases. Both IMD-C outbreaks correlated with a respective PFGE pattern. PFGE analysis demonstrated that the 2004 British Columbia outbreak strain in men who have sex with men was closely related to the 2001 Abbotsford outbreak strain. PorA sequencing data indicated low diversity of class 1 outer membrane proteins in British Columbia, and did not correlate with epidemiological trends. There was a trend for outbreak-associated PFGE types to demonstrate higher case fatality rates.

Catabacter hongkongensis gen. nov., sp. nov., isolated from blood cultures of patients from Hong Kong and Canada.

Four bacterial isolates were recovered from the blood cultures of four patients, two of whom were from Hong Kong and two of whom were from Canada. The two Hong Kong strains were isolated from a 48-year-old man with intestinal obstruction and secondary sepsis (strain HKU16T) and from a 39-year-old man with acute appendicitis (strain HKU17), while the two Canadian strains were isolated from a 74-year-old man with biliary sepsis (strain CA1) and from a 66-year-old woman with metastatic carcinoma and sepsis (strain CA2). While the first three patients survived, the last patient died 2 weeks after the episode of bacteremia. All four isolates are strictly anaerobic, nonsporulating, gram-positive coccobacilli that were unidentified by conventional phenotypic tests and commercial identification systems. They grow on sheep blood agar as nonhemolytic pinpoint colonies after 48 h of incubation at 37 degrees C in an anaerobic environment. All are catalase positive and motile, with flagella. They produce acid from arabinose, glucose, mannose, and xylose. They do not produce indole or reduce nitrate. They are sensitive to penicillin, vancomycin, and metronidazole but resistant to cefotaxime. 16S rRNA gene sequence analysis showed 16.0%, 16.8%, and 21.0% base differences from Clostridium propionicum, Clostridium neopropionicum, and Atopobium minutum, respectively. The G+C content of strain HKU16T is 40.2% +/- 2.2%. Based on their phylogenetic affiliation, unique G+C content, and phenotypic characteristics, we propose a new genus and species, Catabacter hongkongensis gen. nov., sp. nov., to describe the bacterium, for which HKU16 is the type strain, and suggest that it be assigned to a new family, Catabacteriaceae. The gastrointestinal tract was probably the source of the bacterium for at least three of the four patients. The isolation of a catalase-positive, motile, nonsporulating, anaerobic gram-positive bacillus in clinical laboratories should raise the possibility of C. hongkongensis. Further studies should be performed to ascertain the epidemiology and other disease associations of this bacterium.

Direct identification of mycobacteria in primary liquid detection media by partial sequencing of the 65-kilodalton heat shock protein gene.

We investigated extending the use of direct partial hsp65 gene sequencing for the identification of mycobacteria to isolates in primary liquid detection media as an economical, feasible, and more rapid means of identification. During the course of the study, the hsp65 sequence-based identifications for isolates from 670 primary liquid detection media determined to be positive for acid-fast bacilli were compared to the identifications derived from Accuprobes, biochemical test panels, or 16S rRNA gene sequencing. Preliminary analysis indicated a 97.6% concordance, with a final agreement of 99.1% between the identification algorithms. hsp65 sequencing costs (32.84 US dollars) were greater than the cost of identification with Accuprobe (9 US dollars) but less than the cost of the biochemical test panel identification (average cost, 98.90 US dollars) and equivalent to the cost of 16S rRNA sequencing, although there was a referral cost (59.85 US dollars) for the shipping of isolates to another reference laboratory. Analysis indicated that our laboratory would have recognized a cost savings of approximately 12,000 US dollars by using hsp65 sequencing to identify isolates from specimens with a negative fluorescent- smear status and would have achieved further savings by using it as an alternative to biochemical panel testing for fluorescent-smear-positive specimens. The time to identification by hsp65 gene sequencing was slightly longer than that required by the Accuprobe assay (1 versus 2 days), shorter than that required by the biochemical test panels (2 days versus 26 days on average), and more rapid than referral for 16S rRNA gene sequencing.

An Outbreak of Human Coronavirus OC43 Infection and Serological Cross-reactivity with SARS Coronavirus.

BACKGROUND: In summer 2003, a respiratory outbreak was investigated in British Columbia, during which nucleic acid tests and serology unexpectedly indicated reactivity for severe acute respiratory syndrome coronavirus (SARS-CoV). METHODS: Cases at a care facility were epidemiologically characterized and sequentially investigated for conventional agents of respiratory infection, SARS-CoV and other human CoVs. Serological cross-reactivity between SARS-CoV and human CoV-OC43 (HCoV-OC43) was investigated by peptide spot assay. RESULTS: Ninety-five of 142 residents (67%) and 53 of 160 staff members (33%) experienced symptoms of respiratory infection. Symptomatic residents experienced cough (66%), fever (21%) and pneumonia (12%). Eight residents died, six with pneumonia. No staff members developed pneumonia. Findings on reverse transcriptase-polymerase chain reaction assays for SARS-CoV at a national reference laboratory were suspected to represent false positives, but this was confounded by concurrent identification of antibody to N protein on serology. Subsequent testing by reverse transcriptase-polymerase chain reaction confirmed HCoV-OC43 infection. Convalescent serology ruled out SARS. Notably, sera demonstrated cross-reactivity against nucleocapsid peptide sequences common to HCoV-OC43 and SARS-CoV. CONCLUSIONS: These findings underscore the virulence of human CoV-OC43 in elderly populations and confirm that cross-reactivity to antibody against nucleocapsid proteins from these viruses must be considered when interpreting serological tests for SARS-CoV.

Mycobacterium pseudoshottsii sp. nov., a slowly growing chromogenic species isolated from Chesapeake Bay striped bass (Morone saxatilis).

A group of slowly growing photochromogenic mycobacteria was isolated from Chesapeake Bay striped bass (Morone saxatilis) during an epizootic of mycobacteriosis. Growth characteristics, acid-fastness and 16S rRNA gene sequencing results were consistent with those of the genus Mycobacterium. Biochemical reactions, growth characteristics and mycolic acid profiles (HPLC) resembled those of Mycobacterium shottsii, a non-pigmented mycobacterium also isolated during the same epizootic. Sequencing of the 16S rRNA genes, the gene encoding the exported repeated protein (erp) and the gene encoding the 65 kDa heat-shock protein (hsp65) and restriction enzyme analysis of the hsp65 gene demonstrated that this group of isolates is unique. Insertion sequences associated with Mycobacterium ulcerans, IS2404 and IS2606, were detected by PCR. These isolates could be differentiated from other slowly growing pigmented mycobacteria by their inability to grow at 37 degrees C, production of niacin and urease, absence of nitrate reductase, negative Tween 80 hydrolysis and resistance to isoniazid (1 mug ml(-1)), p-nitrobenzoic acid, thiacetazone and thiophene-2-carboxylic hydrazide. On the basis of this polyphasic study, it is proposed that these isolates represent a novel species, Mycobacterium pseudoshottsii sp. nov. The type strain, L15(T), has been deposited in the American Type Culture Collection as ATCC BAA-883(T) and the National Collection of Type Cultures (UK) as NCTC 13318(T).

Rapid and fatal meningococcal disease due to a strain of Neisseria meningitidis containing the capsule null locus.

BACKGROUND: Neisseria meningitidis continues to be an important cause of invasive bacterial disease among children and young adults worldwide. In Canada, N. meningitidis strains that bear serogroups B and C polysaccharide capsules predominate. We report the first documented case of invasive meningococcal disease in an immunocompetent host caused by an acapsular strain of N. meningitidis containing the capsule null locus (cnl). METHODS: Analysis of the isolate was performed with use of serological and molecular methods, including multilocus sequence typing and cnl gene identification. Analysis of 16S ribosomal RNA (rRNA) and porA genes was also performed to confirm the identity of the bacterium. RESULTS: The patient was a healthy, immunocompetent 13-year-old child, and N. meningitidis was recovered from a sample of her cerebrospinal fluid before death. The isolate was nontypeable by both conventional antisera and indirect whole-cell enzyme-linked immuosorbent assay methods using antibodies to serogroups B, C, Y, and W135. The isolate was further identified as a cnl strain, serotype 15 (ST-198). N. meningitidis-specific DNA was identified in the isolate and in the pre- and postmortem specimens by 16S rRNA and porA gene analysis. CONCLUSIONS: This is the first reported case of fatal meningococcal disease caused by an acapsular cnl strain of N. meningitidis that was isolated from an immunocompetent host. Routine molecular diagnostic methods targeted at the cnl locus failed to detect this organism, indicating a need to determine the incidence of infection with cnl strains among patients with culture-negative invasive disease.

Assessment of partial sequencing of the 65-kilodalton heat shock protein gene (hsp65) for routine identification of Mycobacterium species isolated from clinical sources.

We assessed the ability of an in-house database, consisting of 111 hsp65 sequences from putative and valid Mycobacterium species or described groups, to identify 689 mycobacterial clinical isolates from 35 species or groups. A preliminary assessment indicated that hsp65 sequencing confirmed the identification of 79.4% of the isolates from the 32 species examined, including all Mycobacterium tuberculosis complex isolates, all isolates from 13 other species, and 95.6% of all M. avium-M. intracellulare complex isolates. Identification discrepancies were most frequently encountered with isolates submitted as M. chelonae, M. fortuitum, M. gordonae, M. scrofulaceum, and M. terrae. Reexamination of isolates with discrepant identifications confirmed that hsp65 identifications were correct in a further 40 isolates. This brought the overall agreement between hsp65 sequencing and the other identification methods to 85.2%. The remaining 102 isolates had sequence matches below our acceptance criterion, had nondifferential sequence matches between two or more species, were identified by 16S rRNA sequencing as a putative taxonomic group not contained in our database, or were identified by hsp65 and 16S rRNA gene sequencing as a species not in our biochemical test database or had conflicting identifications. Therefore, to incorporate the unconfirmed isolates it was necessary to create 29 additional entries in our hsp65 identification database: 18 associated with valid species, 7 indicating unique sequences not associated with valid or putative species or groups, and 4 associated with unique, but currently described taxonomic groups. Confidence in the hsp65 sequence identification of a clinical isolate is best when sequence matches of 100% occur, but our data indicate that correct identifications can be confidently made when unambiguous matches exceeding 97% occur, but are dependent on the completeness of the database. Our study indicates that for hsp65 sequencing to be an effective means for identifying mycobacteria a comprehensive database must be constructed. hsp65 sequencing has the advantage of being more rapid and less expensive than biochemical test panels, uses a single set of reagents to identify both rapid- and slow-growing mycobacteria, and can provide a more definitive identification.

Use of an internal positive control in a multiplex reverse transcription-PCR to detect West Nile virus RNA in mosquito pools.

We report on the use of West Nile virus Armored RNA as an internal positive control (IPC) for the extraction and reverse transcription-PCR (RT-PCR) of RNA extracted from field-collected mosquitoes and on a multiplex real-time Taqman RT-PCR to simultaneously detect the 3' noncoding region of West Nile virus and the West Nile virus NS5-2 region comprising the IPC. Mosquito pools from the province of British Columbia, Canada (n = 635), were tested in duplicate and found to be negative for West Nile virus and positive for the IPC. Known West Nile virus-positive supernatants from mosquito pools from the provinces of Alberta and Manitoba were tested in duplicate and found to be positive for both regions of the West Nile virus genome. The mean cycle threshold (Ct) value for the IPC in batch extraction controls +/- 2 standard deviations was found to be 36.43 +/- 1.78 cycles. IPCs of 98.4% (624) of West Nile virus-negative pools fell within this range, indicating the reproducibility of RNA extraction and RT-PCR for pools varying in mosquito genus and number. A comparison of mosquito pool genera revealed no significant genus effect on the Ct value of the IPC. The incorporation of West Nile virus Armored RNA as an IPC allows monitoring of RNA extraction and RT-PCR and detection of false-negative results due to failures in these processes or to PCR inhibition, respectively.