Viability testing of material derived from Mycobacterium tuberculosis prior to removal from a Containment Level-III Laboratory as part of a Laboratory Risk Assessment Program

BACKGROUND: In the field of clinical mycobacteriology, Mycobacterium tuberculosis (MTB) can be a difficult organism to manipulate due to the restrictive environment of a containment level 3 (CL3) laboratory. Tests for rapid diagnostic work involving smears and molecular methods do not require CL3 practices after the organism has been rendered non-viable. While it has been assumed that after organism deactivation these techniques can be performed outside of a CL3, no conclusive study has consistently confirmed that the organisms are noninfectious after the theoretical 'deactivation' steps. Previous studies have shown that initial steps (such as heating /chemical fixation) may not consistently kill MTB organisms. METHODS: An inclusive viability study (n = 226) was undertaken to determine at which point handling of culture extraction materials does not necessitate a CL3 environment. Four different laboratory protocols tested for viability included: standard DNA extractions for IS6110 fingerprinting, crude DNA preparations for PCR by boiling and mechanical lysis, protein extractions, and smear preparations. For each protocol, laboratory staff planted a proportion of the resulting material to Bactec 12B medium that was observed for growth for 8 weeks. RESULTS: Of the 208 isolates initially tested, 21 samples grew within the 8-week period. Sixteen (7.7%) of these yielded positive results for MTB that included samples of: deactivated culture resuspensions exposed to 80°C for 20 minutes, smear preparations and protein extractions. Test procedures were consequently modified and tested again (n = 18), resulting in 0% viability. CONCLUSIONS: This study demonstrates that it cannot be assumed that conventional practices (i.e. smear preparation) or extraction techniques render the organism non-viable. All methodologies, new and existing, should be examined by individual laboratories to validate the safe removal of material derived from MTB to the outside of a CL3 laboratory. This process is vital to establish in house biosafety-validated practices with the aim of protecting laboratory workers conducting these procedures

Conventional and molecular epidemiology of Tuberculosis in Manitoba

BACKGROUND: To describe the demographic and geographic distribution of tuberculosis (TB) in Manitoba, thus determining risk factors associated with clustering and higher incidence rates in distinct subpopulations. METHODS: Data from the Manitoba TB Registry was compiled to generate a database on 855 patients with tuberculosis and their contacts from 1992–1999. Recovered isolates of M. tuberculosis were typed by IS6110 restriction fragment length polymorphisms. Bivariate and multivariate logistic regression models were used to identify risk factors involved in clustering. RESULTS: A trend to clustering was observed among the Canadian-born treaty Aboriginal subgroup in contrast to the foreign-born. The dominant type, designated fingerprint type 1, accounts for 25.8% of total cases and 75.3% of treaty Aboriginal cases. Among type 1 patients residing in urban areas, 98.9% lived in Winnipeg. In rural areas, 92.8% lived on Aboriginal reserves. Statistical models revealed that significant risk factors for acquiring clustered tuberculosis are gender, age, ethnic origin and residence. Those at increased risk are: males (p < 0.05); those under age 65 (p < 0.01 for each age subgroup); treaty Aboriginals (p < 0.001), and those living on reserve land (p < 0.001). CONCLUSION: Molecular typing of isolates in conjunction with contact tracing data supports the notion of the largest ongoing transmission of a single strain of TB within the treaty-status population of Canada recorded to date. This data demonstrates the necessity of continued surveillance of countries with low prevalence of the disease in order to determine and target high-risk populations for concentrated prevention and control measures

Low prevalence of gastrointestinal colonization with antimicrobial-resistant bacteria in high risk units in a Canadian tertiary care centre

OBJECTIVE: To determine the prevalence of antimicrobial-resistant bacteria among patients receiving care in high risk units in a Canadian tertiary care centre

Rapid Identification of Fungi by Using the ITS2 Genetic Region and an Automated Fluorescent Capillary Electrophoresis System

Invasive fungal disease often plays an important role in the morbidity and mortality of immunocompromised patients. The poor sensitivity of current fungal blood culture and histological practices has led to the development of highly sensitive and specific molecular techniques, such as the PCR. Sequence variability of the internal transcribed spacer 2 (ITS2) region of fungi is potentially useful in rapid and accurate diagnosis of clinical fungal isolates. PCR with fungus-specific primers targeted toward conserved sequences of the 5.8S and 28S ribosomal DNA (rDNA) results in amplification of the species-specific ITS2 regions, which are variable in amplicon length. We have made use of the ABI PRISM 310 genetic analyzer and the ABI PRISM 310 GeneScan analysis software for the determination of variable size differences of the ITS2 region of clinically important fungi, including Candida and non-Candida yeasts, Aspergillus species, and a variety of dermatophytes. No cross-reaction occurred when samples were tested against human and bacterial genomic DNA. We have found that most clinically significant fungal isolates can be differentiated by this method, and it therefore serves to be a promising tool for the rapid (<7 h) diagnosis of fungemia and other invasive fungal infections