Age differences to methylphenidate-NAc neuronal and behavioral recordings from freely behaving animals.

Methylphenidate (MPD) is a psychostimulant that is widely prescribed to treat attention deficit-hyperactivity disorder, but it is abused recreationally as well. The nucleus accumbens (NAc) is part of the motivation circuit implicated in drug-seeking behaviors. The NAc neuronal activity was recorded alongside the behavioral activity from young and adult rats to determine if there are significant differences in the response to MPD. The same dose of MPD elicits behavioral sensitization in some animals and behavioral tolerance in others. In adult animals, higher doses of MPD resulted in a greater ratio of tolerance/sensitization. Animals who responded to chronic MPD with behavioral sensitization usually exhibited further increases in their NAc neuronal firing rates as well. Different upregulations of transcription factors (ΔFOSB/CREB), variable proportions of D1/D2 dopamine receptors, and modulation from other brain areas may predispose certain animals to express behavioral and neuronal sensitization versus tolerance to MPD.

Pott's puffy tumour caused by mucormycosis.

A case of Pott's puffy tumour in a diabetic patient with renal failure is reported. The patient did not respond to intravenous antibiotics and further investigation revealed that the patient had mucormycosis. As far as we are aware, this is the first case of Pott's puffy tumour due to mucormycosis to be reported in the literature.

Application of mycobacterial interspersed repetitive unit typing to Manitoba tuberculosis cases: can restriction fragment length polymorphism be forgotten?

Since 1993, all Mycobacterium tuberculosis isolates recovered in the province of Manitoba, Canada, have been genotyped by the standard IS6110-restriction fragment length polymorphism (RFLP) method for routine surveillance, prevention, and control purposes. To date, our laboratory has collected 1,290 isolates, from which we have identified approximately 390 unique fingerprint patterns or "types." Although the standard method is well known for being a lengthy and labor-intensive procedure, a more efficient alternative for typing tuberculosis isolates, the mycobacterial interspersed repetitive unit-variable number tandem repeat (MIRU-VNTR) method, has recently gained acceptance. Consequently, all isolates acquired in 2003 (n = 126) were typed by both methods in order to determine the utility of replacing the RFLP method with MIRU typing for all future isolates. Application of Hunter's discriminatory index to the available study population showed that the MIRU method was close in discriminatory power (D) to the RFLP method (D(MIRU) = 0.831 to 0.984 versus D(RFLP) = 0.821 to 0.997). Clustering of isolates by using MIRU data correlated with RFLP-derived clustering, lending useful information for either an investigation or confirmation of an incidence of recent transmission. In addition, it was determined that each predominant RFLP type in Manitoba had a corresponding, recognizable MIRU type. It is conceivable that in the future RFLP typing can be replaced with MIRU for real-time, ongoing tuberculosis surveillance in the province.

Mycobacterium parascrofulaceum sp. nov., novel slowly growing, scotochromogenic clinical isolates related to Mycobacterium simiae.

A group of pigmented, slowly growing mycobacteria identified by 16S rRNA gene sequencing as 'MCRO 33' (GenBank accession no. AF152559) have been isolated from several clinical specimens in various laboratories across Canada. Genotypically, the organism is most closely related to Mycobacterium simiae. However, it presents with a similar phenotypic profile to Mycobacterium scrofulaceum. Several reference strains obtained from ATCC and TMC culture collections, previously identified as M. scrofulaceum or M. simiae, have also been found to possess the MCRO 33 16S rRNA gene sequence. Biochemical testing, susceptibility testing, HPLC, hsp65 gene and 16S-23S spacer (ITS1) sequencing were performed on clinical and reference strains to characterize further this unique species. Of the clinical strains, one was isolated from a cervix biopsy whereas all other clinical isolates were obtained from respiratory samples. In one patient, symptoms, imaging and repeat clinical specimens positive on culture for this organism were suggestive of active clinical disease. The description of this species, for which the name Mycobacterium parascrofulaceum sp. nov. is proposed, follows the present trend of a large number of novel Mycobacterium species identified due in great part to sequence-based methods. The type strain is HSC68T (= ATCC BAA-614T = DSM 44648T).

Strain-related virulence of the dominant Mycobacterium tuberculosis strain in the Canadian province of Manitoba.

SETTING: The Canadian province of Manitoba. OBJECTIVE: To confirm the putative hypervirulence observed in Mycobacterium tuberculosis Type1 strain and further characterize the progression and manifestation of pulmonary tuberculosis caused by this strain in comparison to other common clinical isolates from Manitoba. DESIGN: C3H and BALB/c mice were exposed to aerosols either of Type1, Type2, Type5, Type72 or H37Rv strain to study their respective survival profiles. Additionally, bacillary loads and lung histology were examined at 15 days post-exposure. RESULTS: In both mouse models, Type-1 infected mice succumbed to disease significantly earlier than other strains (p < or =0.0002). Differences between average log(10) CFU values between clinical isolates were less than 1log(10) difference. In C3H mice, the amount of granulomatous inflammation was highest in Type1 infected mice but not significantly different than all clinical strains. In contrast, BALB/c mice infected with Type1 induced the lowest amount of granulomatous inflammation compared to other clinical isolates. CONCLUSION: Our results indicate that although mice infected with the Type1 strain died significantly earlier than mice infected with other clinical strains in both C3H and BALB/c mice, the hypervirulence of the Type1 strain is not attributed to the growth rate of the organism, as differences in growth between clinical M. tuberculosis isolates were insignificant.

Mycobacterium saskatchewanense sp. nov., a novel slowly growing scotochromogenic species from human clinical isolates related to Mycobacterium interjectum and Accuprobe-positive for Mycobacterium avium complex.

A pigmented, slowly growing Mycobacterium avium complex AccuProbe-positive organism was isolated from the sputum and pleural fluid of a 72-year-old female with bronchiectasis. The unusual morphology of the organism prompted further identification by 16S rRNA gene sequencing, revealing a perfect identity with previously uncharacterized strain Mycobacterium sp. MCRO 8 (GenBank accession no. X93034), with the closest established species by 16S rDNA analysis being Mycobacterium interjectum. HPLC of the organism corresponded to previously obtained patterns identified as M. interjectum-like and, upon sequence evaluation of a selection of strains with a similar profile, more were subsequently identified as MCRO 8. A total of 16 strains isolated from human respiratory samples were evaluated in the characterization of this novel species, for which the name Mycobacterium saskatchewanense sp. nov. is proposed. The type strain is strain 00-250(T) (=ATCC BAA-544(T)=DSM 44616(T)=CIP 108114(T)).

Reassessment of sequence-based targets for identification of bacillus species.

The Bacillus genus is a large heterogeneous group in need of an efficient method for species differentiation. To determine the current validity of a sequence-based method for identification and provide contemporary data, PCR and sequencing of a 500-bp product encompassing the V1 to V3 regions of the 16S rRNA gene were undertaken using 65 of the 83 type strains of this genus. This region proved discriminatory between most species (70.0 to 100% similarity), the exceptions being clinically relevant B. cereus and B. anthracis as well as nonpathogenic B. psychrotolerans and B. psychrodurans. Consequently, 27 type and clinical strains from the B. cereus group were used to test alternate targets (rpoB, vrrA, and the 16S-23S spacer region) for identification. The rpoB gene proved the best alternate target, with a conserved 4-nucleotide difference between B. cereus and B. anthracis. The high 16S rRNA gene sequence similarities between some strains demonstrated the need for a polyphasic approach to the systematics of this genus. This approach is one focus of the Ribosomal Differentiation of Medical Microorganisms mandate. Accordingly, the 16S rRNA gene sequences generated in this study have been submitted for inclusion into its publicly accessible, quality-controlled database at http://www.ridom_rdna.de/.

Identification of a predominant isolate of Mycobacterium tuberculosis using molecular and clinical epidemiology tools and in vitro cytokine responses.

BACKGROUND: Tuberculosis (TB) surveillance programs in Canada have established that TB in Canada is becoming a disease of geographically and demographically distinct groups. In 1995, treaty status aboriginals from the province of Manitoba accounted for 46% of the disease burden of this sub-group in Canada. The TB incidence rates are dramatically high in certain reserves of Manitoba and are equivalent to rates in African countries. The objective of our study was to identify prevalent isolates of Mycobacterium tuberculosis in the patient population of Manitoba using molecular epidemiology tools, studying the patient demographics associated with the prevalent strain and studying the in vitro cytokine profiles post-infection with the predominant strain. METHODS: Molecular typing was performed on all isolates available between 1992 to 1997. A clinical database was generated using patient information from Manitoba. THP-1 cells were infected using strains of M. tuberculosis and cytokine profiles were determined using immunoassays for cytokines IL-1beta, IL-10, IL-12, IFN-gamma and TNF-alpha. RESULTS: In Manitoba, 24% of the disease burden is due to a particular M. tuberculosis strain (Type1). The strain is common in patients of aboriginal decent and is responsible for at least 87% of these cases. Cytokine assays indicate that the Type1 strain induces comparatively lower titers of IL-1beta, IFN-gamma and TNF-alpha in infected THP-1 cells as compared to H37Ra and H37Rv strains. CONCLUSION: In Manitoba, Type1 strain is predominant in TB patients. The majority of the cases infected with this particular strain are newly active with a high incidence of respiratory disease, positive chest radiographs and pulmonary cavities. In vitro secretion of IL-1beta, IFN-gamma and TNF-alpha is suppressed in Type1 infected culture samples when compared to H37Ra and H37Rv infected cells.

An outbreak of methicillin resistant Staphylococcus aureus on a burn unit: potential role of contaminated hydrotherapy equipment.

OBJECTIVE: To report a multi-institution outbreak caused by a single strain of methicillin-resistant Staphylococcus aureus (MRSA). OUTBREAK: Between September 19 and November 20, 1996 an index case and five secondary cases of nosocomial MRSA occurred on a 26 bed adult plastic surgery/burn unit (PSBU) at a tertiary care teaching hospital. Between November 11 and December 23, 1996, six additional cases were identified at a community hospital. One of the community hospital cases was transferred from the PSBU. All strains were identical by pulsed-field gel electrophoresis. MRSA may have contributed to skin graft breakdown in one case, and delayed wound healing in others. Patients required 2 to 226 isolation days. CONTROL MEASURES: A hand held shower and stretcher for showering in the hydrotherapy room of the PSBU were culture positive for the outbreak strain, and the presumed means of transmission. Replacement of stretcher showering with bedside sterile burn wound compresses terminated the outbreak. The PSBU was closed to new admissions and transfers out for 11 days during the investigation. Seven of 12 patients had effective decolonization therapy. CONCLUSION: Environmental contamination is a potential source of nosocomial MRSA transmission on a burn unit. Notification among institutions and community care providers of shared patients infected or colonized with an antimicrobial resistant microorganism is necessary.

Necessity of quality-controlled 16S rRNA gene sequence databases: identifying nontuberculous Mycobacterium species.

The use of the 16S rRNA gene for identification of nontuberculous mycobacteria (NTM) provides a faster and better ability to accurately identify them in addition to contributing significantly in the discovery of new species. Despite their associated problems, many rely on the use of public sequence databases for sequence comparisons. To best evaluate the taxonomic status of NTM species submitted to our reference laboratory, we have created a 16S rRNA sequence database by sequencing 121 American Type Culture Collection strains encompassing 92 species of mycobacteria, and have also included chosen unique mycobacterial sequences from public sequence repositories. In addition, the Ribosomal Differentiation of Medical Microorganisms (RIDOM) service has made freely available on the Internet mycobacterial identification by 16S rRNA analysis. We have evaluated 122 clinical NTM species using our database, comparing >1,400 bp of the 16S gene, and the RIDOM database, comparing approximately 440 bp. The breakdown of analysis was as follows: 61 strains had a sequence with 100% similarity to the type strain of an established species, 19 strains showed a 1- to 5-bp divergence from an established species, 11 strains had sequences corresponding to uncharacterized strain sequences in public databases, and 31 strains represented unique sequences. Our experience with analysis of the 16S rRNA gene of patient strains has shown that clear-cut results are not the rule. As many clinical, research, and environmental laboratories currently employ 16S-based identification of bacteria, including mycobacteria, a freely available quality-controlled database such as that provided by RIDOM is essential to accurately identify species or detect true sequence variations leading to the discovery of new species.

Identification of Mycobacterium species by multiple-fluorescence PCR-single-strand conformation polymorphism analysis of the 16S rRNA gene.

Identification of mycobacteria to the species level by growth-based methodologies is a process that has been fraught with difficulties due to the long generation times of mycobacteria. There is an increasing incidence of unusual nontuberculous mycobacterial infections, especially in patients with concomitant immunocompromised states, which has led to the discovery of new mycobacterial species and the recognition of the pathogenicity of organisms that were once considered nonpathogens. Therefore, there is a need for rapid and sensitive techniques that can accurately identify all mycobacterial species. Multiple-fluorescence-based PCR and subsequent single-strand conformation polymorphism (SSCP) analysis (MF-PCR-SSCP) of four variable regions of the 16S rRNA gene were used to identify species-specific patterns for 30 of the most common mycobacterial human pathogens and environmental isolates. The species-specific SSCP patterns generated were then entered into a database by using BioNumerics, version 1.5, software with a pattern-recognition capability, among its multiple uses. Patient specimens previously identified by 16S rRNA gene sequencing were subsequently tested by this method and were identified by comparing their patterns with those in the reference database. Fourteen species whose SSCP patterns were included in the database were correctly identified. Five other test organisms were correctly identified as unique species or were identified by their closest relative, as they were not in the database. We propose that MF-PCR-SSCP offers a rapid, specific, and relatively inexpensive identification tool for the differentiation of mycobacterial species.

Mycobacterium branderi from both a hand infection and a case of pulmonary disease.

Mycobacterium branderi, a potential human pathogen first characterized in 1995, has been isolated from respiratory tract specimens. We report here a case in which M. branderi was the only organism isolated upon culture from a hand infection. This isolate, along with a second isolate from a bronchial specimen, was subjected to conventional identification tests for mycobacterial species. Further analysis by high-performance liquid chromatography (HPLC) of mycolic acids and 16S rRNA gene sequencing was performed, and the antibiotic susceptibility profile was determined for both strains. Biochemical tests and the HPLC pattern were consistent with that of M. branderi and M. celatum, which are very similar. The 16S rRNA gene sequence of both strains corresponded to that of M. branderi and enabled us to confidently differentiate this organism from other closely related species such as M. celatum. This contributes to a further understanding of the status of this species as a potential human pathogen as well as illustrating the need for molecular diagnostics as a complementary method for the identification of rare mycobacterial species.

Evaluation of recA sequences for identification of Mycobacterium species.

16S rRNA sequence data have been used to provide a molecular basis for an accurate system for identification of members of the genus Mycobacterium. Previous studies have shown that Mycobacterium species demonstrate high levels (>94%) of 16S rRNA sequence similarity and that this method cannot differentiate between all species, i.e., M. gastri and M. kansasii. In the present study, we have used the recA gene as an alternative sequencing target in order to complement 16S rRNA sequence-based genetic identification. The recA genes of 30 Mycobacterium species were amplified by PCR, sequenced, and compared with the published recA sequences of M. tuberculosis, M. smegmatis, and M. leprae available from GenBank. By recA sequencing the species showed a lower degree of interspecies similarity than they did by 16S rRNA gene sequence analysis, ranging from 96.2% between M. gastri and M. kansasii to 75.7% between M. aurum and M. leprae. Exceptions to this were members of the M. tuberculosis complex, which were identical. Two strains of each of 27 species were tested, and the intraspecies similarity ranged from 98.7 to 100%. In addition, we identified new Mycobacterium species that contain a protein intron in their recA genes, similar to M. tuberculosis and M. leprae. We propose that recA gene sequencing offers a complementary method to 16S rRNA gene sequencing for the accurate identification of the Mycobacterium species.

Characterization of a toxin A-negative, toxin B-positive strain of Clostridium difficile responsible for a nosocomial outbreak of Clostridium difficile-associated diarrhea.

Clostridium difficile-associated diarrhea (CAD) is a very common nosocomial infection that contributes significantly to patient morbidity and mortality as well as to the cost of hospitalization. Previously, strains of toxin A-negative, toxin B-positive C. difficile were not thought to be associated with clinically significant disease. This study reports the characterization of a toxin A-negative, toxin B-positive strain of C. difficile that was responsible for a recently described nosocomial outbreak of CAD. Analysis of the seven patient isolates from the outbreak by pulsed-field gel electrophoresis indicated that this outbreak was due to transmission of a single strain of C. difficile. Our characterization of this strain (HSC98) has demonstrated that the toxin A gene lacks 1.8 kb from the carboxy repetitive oligopeptide (CROP) region but apparently has no other major deletions from other regions of the toxin A or toxin B gene. The remaining 1.3-kb fragment of the toxin A CROP region from strain HSC98 showed 98% sequence homology with strain 1470, previously reported by M. Weidmann in 1997 (GenBank accession number Y12616), suggesting that HSC98 is toxinotype VIII. The HSC98 strain infecting patients involved in this outbreak produced the full spectrum of clinical illness usually associated with C. difficile-associated disease. This pathogenic spectrum was manifest despite the inability of this strain to alter tight junctions as determined by using in vitro tissue culture testing, which suggested that no functional toxin A was produced by this strain.

Rapid identification of bacteria from positive blood cultures by fluorescence-based PCR-single-strand conformation polymorphism analysis of the 16S rRNA gene.

Bacteremia continues to result in significant morbidity and mortality, particularly in patients who are immunocompromised. Currently, patients with suspected bacteremia are empirically administered broad-spectrum antibiotics, as definitive diagnosis relies upon the use of blood cultures, which impose significant delays in and limitations to pathogen identification. To address the limitations of growth-based identification, the sequence variability of the 16S rRNA gene of bacteria was targeted for rapid identification of bacterial pathogens isolated directly from blood cultures using a fluorescence-based PCR-single-strand conformation polymorphism (SSCP) protocol. Species-specific SSCP patterns were determined for 25 of the most common bacterial species isolated from blood cultures; these isolates subsequently served as a reference collection for bacterial identification for new cases of bacteremia. A total of 272 blood-culture-positive patient specimens containing bacteria were tested. A previously determined SSCP pattern was observed for 251 (92%) specimens, with 21 (8%) specimens demonstrating SSCP patterns distinct from those in the reference collection. Time to identification from blood culture positivity ranged from 1 to 8 days with biochemical testing, whereas identification by fluorescence-based capillary electrophoresis was obtained as early as 7 h at a calculated cost of $10 (U.S. currency) per specimen when tested in batches of 10. Limitations encountered included the inability to consistently detect mixed cultures as well as some species demonstrating identical SSCP patterns. This method can be applied directly to blood cultures or whole-blood specimens, where early pathogen identification would result in a timely diagnosis with possible implications for patient management costs and the mortality and morbidity of infections.

Low prevalence of VRE gastrointestinal colonization of hospitalized patients in Manitoba tertiary care and community hospitals.

OBJECTIVE: To determine the prevalence of vancomycin-resistant enterococci (VRE) bowel colonization in hospitalized patients in Manitoba who had stool specimens collected for Clostridium difficile toxin and/or culture testing. DESIGN: Two tertiary care and five community hospitals in Winnipeg and three rural Manitoba community hospitals participated in this study. From January 1 to December 31, 1997 stool specimens, one per patient, submitted to hospital microbiology laboratories for C difficile toxin and/or culture testing were screened for VRE on colistin-nalidixic acid-vancomycin (6 microg/mL) (CNAV) agar plates. The study was divided into six, eight-week intervals. Stool specimens received in the first two weeks of each eight week interval were screened for VRE. MAIN RESULTS: A total of 1408 stool specimens were submitted over the 48-week study period. Sixty-seven (4.8%) patients with VRE colonization of their lower gastrointestinal tract were identified. Three of the 67 (4.5%) VRE isolates were Enterococcus faecium, with the remaining 64 (95.5%) were Enterococcus gallinarum. The three vancomycin-resistant E faecium -VREF- (from two different Winnipeg hospitals) demonstrated the vanA genotype, and were resistant to vancomycin, teicoplanin and ampicillin. All three VREF isolates also demonstrated high level resistance to both gentamicin and streptomycin but were susceptible to quinuprisitin/dalfopristin and LY333328. CONCLUSION: VRE colonization in hospitalized patients in Manitoba is infrequent and most commonly due to E gallinarum. The prevalence of VREF colonization in the patients studied was 0.2% (three of 1408).

Helicobacter pylori in the Canadian arctic: seroprevalence and detection in community water samples.

OBJECTIVE: Many North American arctic communities are characterized by risk markers associated with Helicobacter pylori (H. pylori) infection, including overcrowded housing and inadequate water supply and sanitation systems. Our aim was to determine the seroprevalence of H. pylori infection in two traditional Inuit communities in the central Canadian arctic and to test for the presence of H. pylori, by polymerase chain reaction (PCR), in local water supplies. METHODS: Samples of venous whole blood from adults and capillary blood from children were collected and analyzed by enzyme immunoassay and Helisal Rapid Test, respectively, for IgG antibody to H. pylori. Antibodies to CagA were detected by enzyme immunoassay, and ABO and Lewis antigens were also determined. Demographic and clinical information were collected by questionnaire. Water samples from each community were tested for H. pylori by PCR. RESULTS: One hundred-thirty (50.8%) of 256 subjects from the two communities were positive for H. pylori IgG antibodies. Seropositive subjects were more likely to be male, compared with seronegative individuals (p = 0.01). Antibody status did not differ with respect to age, community, alcohol or cigarette use, number of persons per household, gastrointestinal complaints or previous investigations, medications, or presence of blood group O, Lewis a-b+. CagA antibodies were detected in 78 (61.9%) of 126 H. pylori-seropositive subjects tested; however, 41 (35.3%) of 116 H. pylori-seronegative subjects were also CagA positive. Water samples taken from the water delivery truck in Chesterfield Inlet and two lakes near Repulse Bay were positive for H. pylori. CONCLUSION: The seroprevalence of H. pylori in the study group was higher than rates in southern Canadian populations, but lower than the seroprevalence previously documented in a Canadian subarctic Indian (First Nations) community. The detection of H. pylori in local water supplies may indicate a natural reservoir for the organism or possible contamination from human sewage.

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.