Biomedical subjects

Christine Y Turenne

Publications and source records attributed to Christine Y Turenne.

10 recordsLinked to original sources

PubMed2006

Evaluation of in situ methods used to detect Mycobacterium avium subsp. paratuberculosis in samples from patients with Crohn's disease.

In common with other diagnostic tests, detection of mycobacteria in tissue by microscopic examination is susceptible to spectrum bias. Since Crohn's disease is defined by the absence of detectable pathogenic organisms, the use of in situ techniques to search for Mycobacterium avium subsp. paratuberculosis in Crohn's disease samples requires validation of methods in a paucibacillary setting. To generate paucibacillary infection, C57BL/6 mice were artificially infected with Mycobacterium avium subsp. paratuberculosis strain K10 and M. tuberculosis H37Rv, yielding tissues harboring fewer than one bacillus per oil immersion field. Serial sections of organs were then studied by cell wall-based staining techniques (Ziehl-Neelsen and auramine rhodamine) and nucleic acid-based staining techniques (in situ hybridization [ISH] and indirect in situ PCR [IS PCR]). Microscopic examination and measurement of morphometric parameters of bacilli revealed that for all methods, Mycobacterium avium subsp. paratuberculosis bacilli were observed to be shorter, smaller, and less rod shaped than M. tuberculosis bacilli. Ziehl-Neelsen, auramine rhodamine stains, ISH targeting rRNA, and IS-PCR targeting the IS900 element afforded comparable sensitivities, but for all methods, visualization of individual bacterial forms required magnification x1,000. Auramine rhodamine staining and IS-PCR generated positive signals in negative controls, indicating the nonspecificity of these assays. Together, our results indicate that detection of Mycobacterium avium subsp. paratuberculosis bacilli in tissue requires oil immersion microscopy, that rRNA-ISH provides sensitivity and specificity comparable to those of Ziehl-Neelsen staining, and that the microscopic detection limit for Mycobacterium avium subsp. paratuberculosis in tissue is governed more by bacterial burden than by staining method.

Animals↗

PubMed2006

Sequencing of hsp65 distinguishes among subsets of the Mycobacterium avium complex.

The Mycobacterium avium complex consists of epidemiologically distinct subsets. The classification of these subsets is complicated by a number of factors, including the ambiguous results obtained with phenotypic and genetic assays and the recent appreciation that human and avian strains appear to be distinct. In previous work, sequencing based on a 441-bp portion of the hsp65 gene has proven to efficiently classify isolates within the Mycobacterium genus but provides low resolution for distinguishing among members of the M. avium complex. Therefore, in this study, we have targeted the more variable 3' region of the hsp65 gene to determine whether it can effectively discriminate M. avium complex isolates at the levels of species and subspecies. Primers designed for this target consistently generated amplicons for all organisms classified as M. avium complex. Sequences obtained indicate that M. intracellulare is genetically divergent from M. avium organisms, and distinct sequevars were obtained for M. avium subsets, including M. avium subsp. avium (bird type), M. avium subsp. hominissuis, and M. avium subsp. paratuberculosis. In addition, sequence differences served to distinguish bovine from ovine strains of M. avium subsp. paratuberculosis. A unique profile for M. avium subsp. silvaticum was not obtained. These results indicate that sequencing the 3' region of the hsp65 gene can simply and unambiguously distinguish species and subspecies of the M. avium complex.

Animals↗

PubMed2006

Insertion sequence IS900 revisited.

Many studies investigating Mycobacterium avium subsp. paratuberculosis in Crohn's disease have used molecular detection of IS900 in clinical samples, but some have described polymorphisms in IS900 as variants of this organism. Analysis of 23 M. avium subsp. paratuberculosis isolates revealed that IS900 is highly conserved, with only two sequevars distinguishing sheep and cattle lineages. Amplification of IS900-like sequences is not sufficient as a proxy for M. avium subsp. paratuberculosis.

Animals↗

PubMed2006

Differentiating host-associated variants of Mycobacterium avium by PCR for detection of large sequence polymorphisms.

The Mycobacterium avium species consists of a group of organisms that are genetically related but phenotypically diverse, with certain variants presenting clear differences in terms of their host association and disease manifestations. The ability to distinguish between these subtypes is of relevance for accurate diagnosis and for control programs. Using a comparative genomics approach, we have uncovered large sequence polymorphisms that are, respectively, absent from bird-type M. avium isolates and from cattle types and sheep types of M. avium subsp. paratuberculosis. By evaluating the distribution of these genomic polymorphisms across a panel of strains, we were able to assign unique genomic signatures to these host-associated variants. We propose a simple PCR-based strategy based on these polymorphisms that can rapidly type M. avium isolates into these subgroups.

Animals↗

PubMed2005

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 degrees 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.

Clinical Laboratory Techniques↗

PubMed2005

Genomic polymorphisms for Mycobacterium avium subsp. paratuberculosis diagnostics.

Mycobacterium avium subsp. paratuberculosis is an emerging pathogen of mammals and is being actively investigated as a possible zoonotic agent. The lack of reliable diagnostic assays has hampered rational assessment of the prevalence of this organism in humans and animals. We have used a comparative genomic approach to reveal genomic differences between M. avium subsp. paratuberculosis and its close relative M. avium subsp. avium, a highly prevalent environmental organism. From computational and DNA microarray-based study of two prototype strains, M. avium subsp. avium strain 104 and M. avium subsp. paratuberculosis strain K10, we have uncovered two types of large sequence polymorphisms (LSPs): those present in the former but missing in the latter (LSP(A)s) and those only present in the latter (LSP(P)s). We examined the distribution of 3 LSP(A)s and 17 LSP(P)s across a panel of 383 M. avium complex isolates in order to determine their potential utility for the development of accurate diagnostic tests. Our results show that the absence of LSP(A)8 is 100% specific for the identification of M. avium subsp. paratuberculosis. Of the 17 LSP(P)s, 10 regions were not specific for M. avium subsp. paratuberculosis while 7 were shown to be highly specific (>98%) and, in some cases, highly sensitive as well (up to 95%). These data highlight the need to evaluate these regions across a diverse panel of clinical and environmental isolates and indicate the LSPs best suited for M. avium subsp. paratuberculosis diagnostics.

Genome, Bacterial↗

PubMed2004

First case report of catheter-related bacteremia due to "Mycobacterium lacticola".

This is the first report of infection caused by "Mycobacterium lacticola," a rapidly growing, scotochromogenic mycobacterium that was isolated from the blood of an immunosuppressed child. The organism was identified by sequence analysis of >1,400 bp of the 16S rRNA gene. The clinical relevance of this isolate, coupled with its unique 16S rRNA gene sequence, should prompt further investigation to establish this organism as a valid mycobacterial species.

Bacteremia↗

PubMed2003

Conventional methods versus 16S ribosomal DNA sequencing for identification of nontuberculous mycobacteria: cost analysis.

The clinical profile of nontuberculous mycobacteria (NTM) has been raised by the human immunodeficiency virus and AIDS pandemic. Different laboratory techniques, often molecular based, are available to facilitate the rapid and accurate identification of NTM. The expense of these advanced techniques has been questioned. At the National Reference Center for Mycobacteriology and the Health Sciences Center, University of Manitoba, in Winnipeg, Canada, we performed a direct cost analysis of laboratory techniques for commercial DNA probe-negative (Gen-Probe, Inc., San Diego, Calif.), difficult-to-identify NTM. We compared the costs associated with conventional phenotypic methodology (biochemical testing, pigment production, growth, and colony characteristics) and genotypic methodology (16S ribosomal DNA [rDNA] sequence-based identification). We revealed a higher cost per sample with conventional methods, and this cost varied with organism characteristics: $80.93 for slowly growing, biochemically active NTM; $173.23 for slowly growing, biochemically inert NTM; and $129.40 for rapidly growing NTM. The cost per sample using 16S rDNA sequencing was $47.91 irrespective of organism characteristics, less than one-third of the expense associated with phenotypic identification of biochemically inert, slow growers. Starting with a pure culture, the turnaround time to species identification is 1 to 2 days for 16S rDNA sequencing compared to 2 to 6 weeks for biochemical testing. The accuracy of results comparing both methodologies is briefly discussed. 16S rDNA sequencing provides a cost-effective alternative in the identification of clinically relevant forms of probe-negative NTM. This concept is not only useful in mycobacteriology but also is highly applicable in other areas of clinical microbiology.

Costs and Cost Analysis↗

PubMed2003

Soft tissue infection caused by a novel pigmented, rapidly growing mycobacterium species.

Molecular techniques are playing an important role in the diagnosis of nontuberculous mycobacterial infections. This case report describes a chronic soft tissue infection in an immunocompetent patient caused by a previously undescribed pigmented, rapidly growing Mycobacterium species, emphasizing the importance of clinical suspicion and effective laboratory techniques in the diagnosis and treatment of infection.

Adult↗

PubMed2003

A high proportion of novel mycobacteria species identified by 16S rDNA analysis among slowly growing AccuProbe-negative strains in a clinical setting.

Sequencing of the 16S ribosomal DNA (rDNA) for identification of nontuberculous mycobacteria (NTM) has contributed to the establishment of more than 35 new species during the last decade. Increasingly, NTM are accepted as potential or proven pathogens. We identified, by 16S rDNA sequence analysis, slowly growing NTM isolates negative by AccuProbe (GenProbe, San Diego, CA) that previously were identified by using conventional biochemical techniques, to determine the accuracy of reporting AccuProbe-negative NTM prior to sequence-based identification. Of 82 strains, 30 were deemed novel. An attempt was made to determine the clinical importance of previously misidentified novel species. Clinical cases are described for a number of strains previously identified as Mycobacterium terrae complex, Mycobacterium scrofulaceum, and Mycobacterium avium complex. As sequence-based identification methods become more commonplace in clinical microbiology laboratories, there is a need to understand the significance of previously undescribed species, which often mimic and subsequently are identified as well-established species.

Adult↗