Frances Jamieson

Genotype and subtype analyses of Cryptosporidium isolates from dairy calves and humans in Ontario

To assess the importance of dairy cattle as a source of human Cryptosporidium infections in Ontario, Canada, 44 Cryptosporidium isolates from neonatal dairy calves and 11 from sporadic human cases of cryptosporidiosis in the province were genotyped by PCR-RFLP analyses of the Cryptosporidium oocyst wall protein (COWP) and 18S rRNA genes. Isolates were also subtyped by sequence analysis of the 60-kDa glycoprotein (GP60) gene. All bovine isolates successfully subtyped belonged to Cryptosporidium parvum subtype family (allele) IIa. Seven subtypes of this family were identified among the bovine isolates. Four human isolates were Cryptosporidium hominis, of alleles Ia, Id, and Ie. Of the remaining seven human specimens, four were C. parvum allele IIa, two were C. parvum of an undetermined subtype, and one was identified as Cryptosporidium cervine genotype. Three of the C. parvum isolates from humans were the same subtypes as isolates from the calves. These findings suggest that cattle and other ruminants may be a source of sporadic human infections in Ontario. This is the first published description of Cryptosporidium genotypes and subtypes in Ontario, and is the second published report of human infection with Cryptosporidium cervine genotype.

Detection of Airborne Severe Acute Respiratory Syndrome (SARS) Coronavirus and Environmental Contamination in SARS Outbreak Units

Abstract Severe acute respiratory syndrome (SARS) is characterized by a risk of nosocomial transmission; however, the risk of airborne transmission of SARS is unknown. During the Toronto outbreaks of SARS, we investigated environmental contamination in SARS units, by employing novel air sampling and conventional surface swabbing. Two polymerase chain reaction (PCR)–positive air samples were obtained from a room occupied by a patient with SARS, indicating the presence of the virus in the air of the room. In addition, several PCR-positive swab samples were recovered from frequently touched surfaces in rooms occupied by patients with SARS (a bed table and a television remote control) and in a nurses’ station used by staff (a medication refrigerator door). These data provide the first experimental confirmation of viral aerosol generation by a patient with SARS, indicating the possibility of airborne droplet transmission, which emphasizes the need for adequate respiratory protection, as well as for strict surface hygiene practices

Increased Burden of Illness Associated with Antimicrobial-Resistant Salmonella enterica Serotype Typhimurium Infections

This study investigated the burden of illness associated with 440 cases of Salmonella enterica serotype Typhimurium infection identified in Canada between December 1999 and November 2000. We categorized case subjects infections by definitive phage type 104 (DT104) and antimicrobial-resistance patterns. These variables were then investigated as risk factors for hospitalization. Hospitalization was more likely to occur among case subjects whose infections were resistant to at least ampicillin, chloramphenicol and/or kanamycin, streptomycin, sulphamethoxazole, and tetracycline (R-type AK/CSSuT; odds ratio [OR], 2.3; P=.003), compared with case subjects with AK/CSSuT-susceptible infections, and among case subjects with non-DT104 R-type AKSSuT infections (OR, 3.6; P=.005), compared with case subjects with non-DT104 AKSSuT-susceptible infections. In contrast, hospitalization rates did not differ between case subjects with DT104 infections and case subjects with non-DT104 infections or between case subjects with DT104 R-type ACSSuT infections and case subjects with DT104 ACSSuT-susceptible infections. We estimated that 57% of the hospitalizations among AK/CSSuT case subjects and 72% of the hospitalizations among non-DT104 AKSSuT case subjects were attributable to the resistance patterns of the infections.

Onychomycosis: a critical study of techniques and criteria for confirming the etiologic significance of nondermatophytes

Nondermatophytic filamentous fungi (NDF) other than Scytalidium species are variously said to cause between 0 and 50% of all toenail onychomycoses, though most estimates are in the 2-5% range. Due to the difficulty of obtaining gold standard control data for comparison, the accuracy of many laboratory evaluation processes used to deal with potential NDF onychomycoses has never been rigorously measured, thus allowing use of differing interpretations of the significance of cultures. To allow valid comparison of these procedures and interpretations, a large series of patients who declined treatment were sampled on multiple occasions from all apparently onychomycotic toenails until adequate certainty had been attained that all etiologic agents had been isolated and, where necessary, confirmed as etiologic via consistent repeated isolation. This information was used to evaluate results that had been obtained in the initial direct microscopy and culture studies for the same patient population, as such results are strongly relied on in routine diagnosis. Direct microscopy (KOH) was found to be 73.8% sensitive for dermatophytes and 67.2% sensitive for proven etiologic NDF (difference not significant). Culture of the initial specimen coincidentally had a sensitivity of 74.6% for both fungal groups. KOH and culture in tandem were 83.9% sensitive for indicating fungal etiology based on the first specimen. Different evaluative frameworks currently used to interpret NDF isolations were contrasted. The classic evaluation procedure, in which all NDF considered etiologic must be isolated from at least two successive nail specimens, at least one of which must be microscopic positive for compatible fungal filaments, had a sensitivity of 59.5% but a specificity of 100% for true NDF infections and mixed NDF/dermatophyte infections. The most widely used simple association evaluation criterion, in which NDF outgrowth is considered etiologic whenever direct microscopy is positive for fungal elements and no dermatophyte grows out from the initial specimen, had a sensitivity of 53.6% and a specificity of 70.3% for NDF infections. A frequently criticized, but in some quarters still advocated, variant of the simple association criterion considers NDF outgrowth to be significant whenever the corresponding specimen is positive for fungal filaments in direct microscopy; application of this criterion yielded a sensitivity of 60.7% for true infections but a specificity of only 42%. With the aid of two standard notes soliciting repeat specimens, the classic criterion was able to attain 92.7% accuracy in recognizing all NDF etiologic agents and 100% accuracy in disregarding all contaminants from initial specimens that were positive in direct microscopy and yielded a filamentous fungus in initial culture. Even in exhaustive longitudinal study, only 20.2% of NDF infections were found to be associated with a concurrent dermatophytosis. In auxiliary studies, some nails remained NDF-infected after dermatophytes had been successfully eliminated by therapy.

Molecular typing of a Legionella pneumophila outbreak in Ontario, Canada

An outbreak of Legionnaires disease at a long-term care facility in Ontario, Canada from September to October 2005 resulted in the death of 23 residents and the illness of 112 other people. In response, molecular methods were developed to detect Legionella pneumophila in clinical lung samples and to subtype isolates from clinical and environmental samples. The targeted genetic loci included Legionella-specific virulence determinants (mip, icmO, sidA and lidA) and core bacterial determinants (ftsZ, trpS and dnaX). An established amplified fragment length polymorphism typing method provided the first indication of genetic relatedness between strains recovered from clinical samples and strains cultured from environmental samples taken from the outbreak site. These associations were verified using the European Working Group for Legionella Infections sequence-based typing protocol targeting the flaA, pilE, asd, mip, mompS and proA loci. These molecular typing methods confirmed the outbreak source as a contaminated air conditioning cooling tower.

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.

Limited genetic diversity in Salmonella enterica Serovar Enteritidis PT13

BackgroundSalmonella enterica serovar Enteritidis has emerged as a significant foodborne pathogen throughout the world and is commonly characterized by phage typing. In Canada phage types (PT) 4, 8 and 13 predominate and in 2005 a large foodborne PT13 outbreak occurred in the province of Ontario. The ability to link strains during this outbreak was difficult due to the apparent clonality of PT13 isolates in Canada, as there was a single dominant pulsed-field gel electrophoresis (PFGE) profile amongst epidemiologically linked human and food isolates as well as concurrent sporadic strains. The aim of this study was to perform comparative genomic hybridization (CGH), DNA sequence-based typing (SBT) genomic analyses, plasmid analyses, and automated repetitive sequence-based PCR (rep-PCR) to identify epidemiologically significant traits capable of subtyping S. Enteritidis PT13.ResultsCGH using an oligonucleotide array based upon chromosomal coding sequences of S. enterica serovar Typhimurium strain LT2 and the Salmonella genomic island 1 successfully determined major genetic differences between S. Typhimurium and S. Enteritidis PT13, but no significant strain-to-strain differences were observed between S. Enteritidis PT13 isolates. Individual loci (safA and fliC) that were identified as potentially divergent in the CGH data set were sequenced in a panel of S. Enteritidis strains, and no differences were detected between the PT13 strains. Additional sequence-based typing was performed at the fimA, mdh, manB, cyaA, citT, caiC, dmsA, ratA and STM0660 loci. Similarly, no diversity was observed amongst PT13 strains. Variation in plasmid content between PT13 strains was observed, but macrorestriction with Bgl II did not identify further differences. Automated rep-PCR patterns were variable between serovars, but S. Enteritidis PT13 strains could not be differentiated.ConclusionNone of the methods identified any significant variation between PT13 strains. Greater than 11,300 base pairs of sequence for each of seven S. Enteritidis PT13 strains were analyzed without detecting a single polymorphic site, although diversity between different phage types of S. Enteritidis was observed. These data suggest that Canadian S. Enteritidis PT13 strains are highly related genetically.

Nosocomial Transmission of Congenital Tuberculosis in a Neonatal Intensive Care Unit

Congenital tuberculosis is uncommon, and nosocomial transmission from a congenitally infected infant to another infant has not been reported in the English literature. We report an investigation of 2 infants with tuberculosis who were cared for in the same neonatal intensive care unit. Isolates from both infants were genetically indistinguishable. Transmission between the 2 infants was likely due to contaminated respiratory equipment.

Comparison of in-house and commercial 16S rRNA sequencing with high-performance liquid chromatography and genotype AS and CM for identification of nontuberculous mycobacteria

Sequencing of the 16S gene or other targets and line probe assay are in wide use for the identification of nontuberculous mycobacteria. We compared in-house and commercial sequencing with 3 sequence databases against high-performance liquid chromatography (HPLC) and line probe assay (HAIN Genotype AS and CM) for the identification of 84 reference, clinical, and unique strains representing 41 species. Consensus of methods was used as reference standard. Sequencing identification was more specific and flexible than HPLC, but it was limited by database content and quality as well as fragment length. No one database satisfied all requirements. In-house sequencing was lower in cost than commercial sequencing or line probe assay.

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.