Rapid detection of meticillin-resistant Staphylococcus aureus bacteraemia using combined three-hour short-incubation matrix-assisted laser desorption/ionization time-of-flight MS identification and Alere Culture Colony PBP2a detection test.

Meticillin-resistant Staphylococcus aureus (MRSA) bloodstream infection is responsible for significant morbidity, with mortality rates as high as 60 % if not treated appropriately. We describe a rapid method to detect MRSA in blood cultures using a combined three-hour short-incubation BRUKER matrix-assisted laser desorption/ionization time-of-flight MS BioTyper protocol and a qualitative immunochromatographic assay, the Alere Culture Colony Test PBP2a detection test. We compared this combined method with a molecular method detecting the nuc and mecA genes currently performed in our laboratory. One hundred and seventeen S. aureus blood cultures were tested of which 35 were MRSA and 82 were meticillin-sensitive S. aureus (MSSA). The rapid combined test correctly identified 100 % (82/82) of the MSSA and 85.7 % (30/35) of the MRSA after 3 h. There were five false negative results where the isolates were correctly identified as S. aureus, but PBP2a was not detected by the Culture Colony Test. The combined method has a sensitivity of 87.5 %, specificity of 100 %, a positive predictive value of 100 % and a negative predictive value of 94.3 % with the prevalence of MRSA in our S. aureus blood cultures. The combined rapid method offers a significant benefit to early detection of MRSA in positive blood cultures.

Rapid fecal calprotectin testing to assess for endoscopic disease activity in inflammatory bowel disease: A diagnostic cohort study.

BACKGROUND AND AIM: With increasing numbers of patients diagnosed with inflammatory bowel disease (IBD), it is important to identify noninvasive methods of detecting disease activity. The aim of this study is to examine the diagnostic accuracy of fecal rapid calprotectin (FC) testing in the detection of endoscopically active IBD. PATIENTS AND METHODS: All consecutive patients presenting to outpatient clinics with lower gastrointestinal symptoms were prospectively recruited. Patients provided FC samples. Sensitivity (Sn), specificity (Sp), positive predictive value (PPV), and negative predictive value (NPV) for FC were calculated. Receiver-operator characteristics (ROC) curve was used to identify the ideal FC cutoff that predicts endoscopic disease activity. Correlation between FC and endoscopic disease activity, disease location, and C-reactive protein (CRP) levels were measured. RESULTS: One hundred and twenty-six patients, of whom 52% were females, were included in the final analysis with a mean age of 44.4 ± 16.7 years. Comparing FC to endoscopic findings, the following results were calculated: A cutoff point of 100 µg/g showed Sn = 83%, Sp = 67%, PPV = 65%, and NPV = 85%; and 200 µg/g showed Sn = 66%, Sp = 82%, PPV = 73%, and NPV = 77%. Based on ROC curve, the best FC cutoff point to predict endoscopic disease activity was 140 µg/g. Using this reference, FC levels strongly correlated with colorectal, ileocolonic, and ileal disease and predicted endoscopic activity. CONCLUSIONS: FC is an accurate test when used as an initial screening tool for patients suspected of having active IBD. Given its noninvasive nature, it may prove to reduce the need for colonoscopy and be an added tool in the management of IBD.

MRJP1-containing glycoproteins isolated from honey, a novel antibacterial drug candidate with broad spectrum activity against multi-drug resistant clinical isolates.

The emergence of extended- spectrum ß-lactamase (ESBL) is the underlying cause of growing antibiotic resistance among Gram-negative bacteria to ß-lactam antibiotics. We recently reported the discovery of honey glycoproteins (glps) that exhibited a rapid, concentration-dependent antibacterial activity against both Gram-positive Bacillus subtilis and Gram-negative Escherichia coli that resembled action of cell wall-active ß-lactam drugs. Glps showed sequence identity with the Major Royal Jelly Protein 1 (MRJP1) precursor that harbors three antimicrobial peptides: Jelleins 1, 2, and 4. Here, we used semi-quantitative radial diffusion assay and broth microdilution assay to evaluate susceptibility of a number of multi-drug resistant (MDR) clinical isolates to the MRJP1-contaning honey glycoproteins. The MDR bacterial strains comprised three methicillin-resistant Staphylococcus aureus (MRSA), four Pseudomonas aeruginosa, two Klebsiella pneumoniae, two vancomycin-resistant Enterococci (VRE), and five ESBL identified as one Proteus mirabilis, three E. coli, and one E. coli NDM. Their resistance to different classes of antibiotics was confirmed using automated system Vitek 2. MDR isolates differed in their susceptibility to glps with MIC90 values ranging from 4.8 µg/ml against B. subtilis to 14.4 µg/ml against ESBL K. pneumoniae, Klebsiella spp. ESBL and E. coli and up to 33 µg/ml against highly resistant strains of P. aeruginosa. Glps isolated from different honeys showed a similar ability to overcome bacterial resistance to ß-lactams suggesting that (a) their mode of action is distinct from other classes of ß-lactams and that (b) the common glps structure was the lead structure responsible for the activity. The results of the current study together with our previous evidence of a rapid bactericidal activity of glps demonstrate that glps possess suitable characteristics to be considered a novel antibacterial drug candidate.

Determination of real-time polymerase chain reaction uncertainty of measurement using replicate analysis and a graphical user interface with Fieller's theorem.

BACKGROUND: Disease monitoring of viruses using real-time polymerase chain reaction (PCR) requires knowledge of the precision of the test to determine what constitutes a significant change. Calculation of quantitative PCR confidence limits requires bivariate statistical methods. OBJECTIVE: To develop a simple-to-use graphical user interface to determine the uncertainty of measurement (UOM) of BK virus, cytomegalovirus (CMV) and Epstein-Barr virus (EBV) real-time PCR assays. METHODS: Thirty positive clinical samples for each of the three viral assays were repeated once. A graphical user interface was developed using a spreadsheet (Excel, Microsoft Corporation, USA) to enable data entry and calculation of the UOM (according to Fieller's theorem) and PCR efficiency. RESULTS: The confidence limits for the BK virus, CMV and EBV tests were ∼0.5 log, 0.5 log to 1.0 log, and 0.5 log to 1.0 log, respectively. The efficiencies of these assays, in the same order were 105%, 119% and 90%. The confidence limits remained stable over the linear range of all three tests. DISCUSSION: A >5 fold (0.7 log) and a >3-fold (0.5 log) change in viral load were significant for CMV and EBV when the results were ≤1000 copies/mL and >1000 copies/mL, respectively. A >3-fold (0.5 log) change in viral load was significant for BK virus over its entire linear range. PCR efficiency was ideal for BK virus and EBV but not CMV. Standardized international reference materials and shared reporting of UOM among laboratories are required for the development of treatment guidelines for BK virus, CMV and EBV in the context of changes in viral load.

HISTORIQUE: Pour surveiller les virus au moyen de la réaction en chaîne de la polymérase (PCR) en temps réel, il faut connaître la précision du test pour déterminer ce qui constitue un changement important. Il faut des méthodes statistiques bivariées pour calculer les limites de confiance de la PCR quantitative. OBJECTIF: Élaborer une interface utilisateur graphique facile à utiliser pour déterminer l'incertitude des mesures (IDM) du virus BK, du cytomégalovirus (CMV) et du virus d'Epstein-Barr (VEB) par PCR en temps réel. MÉTHODOLOGIE: Trente échantillons cliniques positifs de cha-cune des analyses virales ont été répétés une fois. Une interface utilisateur graphique a été élaborée au moyen d'un chiffrier (Excel, Microsoft Corporation, États-Unis) pour saisir les données et calculer l'IDM (selon le théorème de Fieller) et l'efficacité de la PCR. RÉSULTATS: Les limites de confiance des tests du virus BK, du CMV et du VEB étaient de ∼0,5 log, 0,5 log à 1,0 log, et 0,5 log à 1,0 log, respectivement. L'efficacité de ces analyses était de 105 %, de 119 % et de 90 %, respectivement. Les limites de confiance sont demeurées stables pendant la trajectoire linéaire de ces trois tests. EXPOSÉ: Un changement de la charge virale plus de cinq fois plus élevé (0,7 log) et plus de trois fois plus élevé (0,5 log) était important pour le CMV et le VEB lorsque les résultats étaient d'un maximum de 1 000 copies/mL et de plus de 1 000 copies/mL, respectivement. Un changement de la charge virale plus de trois fois élevé (0,5 log) était important pour toute la trajectoire linéaire du virus BK. La PCR avait une efficacité idéale pour le virus BK et le VEB, mais pas pour le CMV. Il faudra élaborer des normes de référence internationales standardisées et partager les signalements d'IDM entre laboratoires pour préparer des directives thérapeutiques relatives au virus BK, au CMV et au VEB dans le contexte des changements de la charge virale.

Association of CPAP bacterial colonization with chronic rhinosinusitis.

STUDY OBJECTIVE: The purpose of our study was to investigate whether bacterial colonization of the continuous positive air-way pressure (CPAP) machine reservoirs occurred, and if so, if it was related to the development of chronic rhinosinusitis (CRS). DESIGN: Prospective cohort study. SETTING: London Health Sciences Center (LHSC). PATIENTS: Regular CPAP users with obstructive sleep apnea (OSA). INTERVENTIONS: N/A. MEASUREMENTS AND RESULTS: Patient demographics were recorded and they were asked to fill out the chronic sinusitis survey (CSS) form. Patients then had their CPAP machines swabbed. An ANOVA was used to determine if the presence of microbacterial colonization was related to CSS scores. In total, 72 patients were included in the study. There was no significant difference in any of the scores between the group with positive cultures and the group without positive cultures. CONCLUSIONS: Having a positive culture in the cpap reservoir does not seem to lead to an increased symptomatology of crs: although the reservoirs often become colonized, there seems to be no clinical impact.

Nonutility of routine testing of stool for ova and parasites in a tertiary care Canadian centre.

BACKGROUND: In many clinical situations, stool examinations for ova and parasites (O&P) are routine in the work-up of patients with acute or chronic diarrhea. Frequently, these tests are found to be negative for pathogens. The purpose of this study was to examine the diagnostic yield of routine stool testing for O&P in a Canadian tertiary care centre and to estimate the potential clinical benefit of a positive result. PATIENTS AND METHODS: All stool samples sent to the central microbiology laboratory at London Health Sciences Centre were reviewed over a 5-year period ending January 2010. Initial screening was done by direct antigen testing using an enzyme immunoassay (EIA) technique followed by direct microscopy for negative results where there was a high index of suspicion and for positive results to rule out any concurrent parasites not included in the EIA kit. Pathogens identified were categorized and their potential susceptibility to metronidazole was estimated. No clinical data were available, as this was purely a utilization study. RESULTS: A total of 5812 stool tests were ordered. Of these, 5681 (97.7%) were completed. The most common reasons for an incomplete test were sample leakage (n = 38) and use of the incorrect collection kit (n = 32). Direct microscopy identified white blood cells in 17% of patients with positive testing. The most common pathogen was Giardia lamblia , which was detected in 45/83 (54%) of positive specimens. Entamoeba histolytica/Entamoeba dispar was identified in 16/83 (19%) and Cryptosporidium spp. in 10/83 (12%) of positive specimens. Microorganisms not thought to be pathogenic were identified in 7/83 (8%). Direct laboratory costs independent of labor were estimated at $1836 per clinically significant organism identified. Of the 77 specimens positive for pathogenic organisms, 62 (81%) were likely to be sensitive to treatment with metronidazole. CONCLUSION: In a tertiary care centre, the diagnostic yield of routine testing of stool for O&P during the evaluation of patients with acute or chronic diarrhea is low. Most clinically significant positive results should be responsive to metronidazole, but empirical treatment is not encouraged. Strategies to identify patients with a higher likelihood of harboring pathogenic parasites and consideration of empiric metronidazole therapy for patients at highest risk merit further research.

Mechanism of Honey Bacteriostatic Action Against MRSA and VRE Involves Hydroxyl Radicals Generated from Honey's Hydrogen Peroxide.

It has been recently reported that honey hydrogen peroxide in conjunction with unknown honey components produced cytotoxic effects resulting in bacterial growth inhibition and DNA degradation. The objective of this study was twofold: (a) to investigate whether the coupling chemistry involving hydrogen peroxide is responsible for a generation of hydroxyl radicals and (b) whether (•)OH generation affects growth of multi-drug resistant clinical isolates. The susceptibility of five different strains of methicillin-resistant Staphylococcus aureus (MRSA) and four strains of vancomycin-resistant Enterococcus faecium (VRE) isolates from infected wounds to several honeys was evaluated using broth microdilution assay. Isolates were identified to genus and species and their susceptibility to antibiotics was confirmed using an automated system (Vitek(®), Biomérieux(®)). The presence of the mec(A) gene, nuc gene and van(A) and (B) genes were confirmed by polymerase chain reaction. Results showed that no clinical isolate was resistant to selected active honeys. The median difference in honeys MICs against these strains ranged between 12.5 and 6.25% v/v and was not different from the MIC against standard Escherichia coli and Bacillus subtilis. Generation of (•)OH during bacteria incubation with honeys was analyzed using 3'-(p-aminophenyl) fluorescein (APF) as the (•)OH trap. The (•)OH participation in growth inhibition was monitored directly by including APF in broth microdilution assay. The growth of MRSA and VRE was inhibited by (•)OH generation in a dose-dependent manner. Exposure of MRSA and VRE to honeys supplemented with Cu(II) augmented production of (•)OH by 30-fold and increased honey bacteriostatic potency from MIC(90) 6.25 to MIC(90)< 0.78% v/v. Pretreatment of honeys with catalase prior to their supplementation with Cu ions fully restored bacterial growth indicating that hydroxyl radicals were produced from H(2)O(2) via the Fenton-type reaction. In conclusion, we have demonstrated for the first time that bacteriostatic effect of honeys on MRSA and VRE was dose-dependently related to generation of (•)OH from honey H(2)O(2).

Comparison of three phenotypic techniques for detection of methicillin resistance in Staphylococcus spp. reveals a species-dependent performance.

OBJECTIVES: To evaluate the usefulness of the cefoxitin screen in Vitek 2 Gram-positive panels for recognizing methicillin-resistant strains of staphylococci. METHODS: Seven hundred and ninety-nine non-duplicate isolates of Staphylococcus aureus and coagulase-negative strains were included in the study. Methicillin resistance was measured using PCR for the mecA gene, the CLSI cefoxitin disc diffusion method, the Vitek 2 cefoxitin screen and the Vitek 2 oxacillin susceptibility test. RESULTS: Compared with the molecular detection of methicillin resistance the overall sensitivities and specificities of the phenotypic tests for cefoxitin disc diffusion were 94.9% and 97.0%, for Vitek 2 cefoxitin screen were 94.6% and 93.5% and for Vitek 2 oxacillin susceptibility test were 93.8% and 77.9%. The cephamycin tests (cefoxitin disc diffusion and Vitek 2 screen) were not able to identify mecA-positive strains of Staphylococcus simulans. In addition, the performance of the Vitek 2 system was poor against Staphylococcus cohnii subspecies, Staphylococcus hominis hominis and Staphylococcus saprophyticus. CONCLUSIONS: Overall, the performance of the Vitek 2 system for differentiating mecA-positive staphylococci was comparable to PCR and the CLSI disc diffusion method; however, performance was species-dependent. Thus, before accepting the results produced by Vitek 2, species identification may be required.

Prospective comparison of a new chromogenic medium, MRSASelect, to CHROMagar MRSA and mannitol-salt medium supplemented with oxacillin or cefoxitin for detection of methicillin-resistant Staphylococcus aureus.

MRSASelect agar was compared to CHROMagar, mannitol-salt agar with oxacillin, and mannitol-salt agar with cefoxitin (MSA-CFOX) for the isolation of methicillin-resistant Staphylococcus aureus (MRSA). The sensitivities and specificities were 97.3% and 99.8%, 82.9% and 99.1%, 80.2% and 79%, and 99.1% and 84.8%, respectively. MSA-CFOX and MRSASelect had a high sensitivity. MRSASelect, however, was more specific and proved to be a more reliable and rapid medium for the detection of MRSA.

Linking health and ecology in the medical curriculum.

Human health vulnerabilities to ecosystem degradation are well documented. Destabilization of natural ecosystems and the biosphere have posed an entirely new set of risks to human health and preclude any simple extrapolations from the past. Newly emerging diseases, increasing prevalence of many vector borne diseases, increased exposure to harmful UV radiation and a number of other transformations in the natural environment, have decidedly negative implications for the sustainability of human health. Curricula in medical schools are responding to these new realities by exposing the connections between health and ecology. The program in Ecosystem Health at the University of Western Ontario serves as one model for connecting these disciplines. This program has resulted in a perceptible shift in values and professional responsibilities of emerging physicians.