High-order wavefront aberrations due to a laser-induced breakdown spark in still air.

Experimental measurements of the wavefronts of the light from a laser-induced breakdown (LIB) spark in stationary air are presented for the full range of angles between the LIB laser and wavefront sensor up to 90°. The wavefront data are compared to measurements of the spark shape and position acquired from simultaneous photographic images of the spark. The results show that the aberrations from the LIB spark appear primarily as two modes determined from proper orthogonal decomposition, with the amplitude and importance of the modes depending on system parameters and viewing angle. Data are also presented demonstrating the link between the spark wavefront and the spark shape, and conclusions are drawn regarding how to minimize the spark aberrations for optical systems in which the spark is used as a light source to measure optical wavefronts.

Canadian recommendations for laboratory interpretation of multiple or extensive drug resistance in clinical isolates of Enterobacteriaceae, Acinetobacter species and Pseudomonas aeruginosa.

The goal of this document was to provide Canadian laboratories with a framework for consistent reporting and monitoring of multidrug resistant organisms (MDRO) and extensively drug resistant organisms (XDRO) for common gram-negative pathogens. This is the final edition of the interim recommendations, which were modified after one year of broad consultative review. This edition represents a consensus of peer-reviewed information and was co-authored by the Canadian Public Health Laboratory Network and the Canadian Association of Clinical Microbiology and Infectious Diseases. There are two main recommendations. The first recommendation provides standardized definitions for MDRO and XDRO for gram-negative organisms in clinical specimens. These definitions were limited to antibiotics that are commonly tested clinically and, to reduce ambiguity, resistance (rather than non-susceptibility) was used to calculate drug resistance status. The second recommendation identifies the use of standardized laboratory reporting of organisms identified as MDRO or XDRO. Through the broad consultation, which included public health and infection prevention and control colleagues, these definitions are ready to be applied for policy development. Both authoring organizations intend to review these recommendations regularly as antibiotic resistance testing evolves in Canada.

Multilaboratory Evaluation of In Vitro Antifungal Susceptibility Testing of Dermatophytes for ME1111.

ME1111 is a novel small molecule antifungal agent under development for the topical treatment of onychomycosis. Standardization of the susceptibility testing method for this candidate antifungal is needed. Toward this end, 8 independent laboratories determined the interlaboratory reproducibility of ME1111 susceptibility testing. In addition, we subsequently identified 2 strains as quality control (QC) isolates for the method. In the reproducibility study, 5 blinded clinical strains each of Trichophyton rubrum, Trichophyton mentagrophytes, and Epidermophyton floccosum were tested, while the QC study tested 6 blinded T. rubrum or T. mentagrophytes ATCC strains. Testing was performed in frozen microtiter panels according to the Clinical and Laboratory Standards Institute (CLSI) M38-A2 methodology. In the reproducibility study, 9 of 15 clinical strains showed interlaboratory agreement of >90% at the 80% inhibition endpoint, with a range of agreement of 76.2% to 100%. In the QC study, 4 of the 6 ATCC strains showed interlaboratory agreement of >90%. ME1111 demonstrated excellent interlaboratory agreement when tested against dermatophytes. Based on this data, the CLSI Subcommittee on Antifungal Susceptibility Tests approved the susceptibility testing of ME1111 against dermatophytes according to M38-A2 methodology, which stipulates RPMI 1640 as the test medium, an inoculum size of 1 to 3 × 10(3) CFU/ml, and an incubation time and temperature of 96 h at 35°C. The MIC endpoint should be 80% inhibition compared with the growth control. T. rubrum ATCC MYA-4438 and T. mentagrophytes ATCC 28185 were selected as QC isolates, with an acceptable range of 0.12 to 1 µg/ml for the two strains.

The management of upper respiratory tract infections.

Upper respiratory tract infections (URTIs), generally termed colds, sore throats and coughs, are common presentations in primary care. This article discusses the clinical picture, management, significant differential diagnosis, and specifically, when antibiotics may be required for an URTI.

Wavefront measurements of a laser-induced breakdown spark in still air.

Experimental measurements of the wavefronts of the light from a laser-induced breakdown (LIB) spark in non-moving air are presented and compared to spark dimensional data acquired from photographic measurements of the spark. The data show that the variation in the spark emitted wavefront between ignitions can be directly related to the motion of the spark volumetric centroid. The dominant modal components of the emitted wavefront variations are presented, as well as quantitative results for the magnitude of the wavefront variations. The results are relevant to the use of LIB as a light source for the measurement of optical aberrations such as those caused by compressible (i.e., "aero-optic") flows around an aircraft in flight, and data are shown indicating that LIB could be successfully used to measure the aberrating effect of compressible shear layers and boundary layers at typical cruise Mach numbers.

First comprehensive evaluation of the M.I.C. evaluator device compared to Etest and CLSI reference dilution methods for antimicrobial susceptibility testing of clinical strains of anaerobes and other fastidious bacterial species.

The new M.I.C. Evaluator strip uses test methodology and the recording of results that are similar to those of Etest. For this first assessment, 102 clinical strains of anaerobic bacteria from 12 genera and 155 strains from 7 genera and 8 species of fastidious bacteria were tested by M.I.C. Evaluator, Etest, and agar dilution or broth microdilution as a reference standard. Ampicillin, amoxicillin, amoxicillin-clavulanate, cefotaxime, ciprofloxacin, erythromycin, imipenem, levofloxacin, metronidazole, penicillin, and tetracycline were tested depending on the species. Agar dilution for anaerobes was performed according to CLSI document M11-A7. For the fastidious bacteria, CLSI document M45-A2 was followed. For the anaerobes, essential and categorical agreement between M.I.C. Evaluator and Etest was >90%. Compared to agar dilution, essential agreement was low for both strip tests, and many very major errors were observed for metronidazole (13 to 14%) and penicillin (8 to 9%) with isolates from the Bacteroides fragilis group and Clostridium species. For fastidious species, essential agreements for M.I.C. Evaluator and Etest plus or minus one doubling dilution were >95%. Compared to broth microdilution, essential agreements were low (40 to 90%) plus or minus one dilution and were >90% plus or minus two dilutions, with high overall category agreement (CA). Major and minor errors were within established parameters for all strains tested. The M.I.C. Evaluator strips were equivalent to Etest for anaerobes and fastidious species. These observations require further investigation to determine which methods provide the most accurate MIC for clinical utility. The further evaluation of additional M.I.C. Evaluator agents will be performed as they become available.

First comprehensive evaluation of the M.I.C. evaluator device compared to Etest and CLSI broth microdilution for MIC testing of aerobic Gram-positive and Gram-negative bacterial species.

The M.I.C. Evaluator strip (Thermo Fisher Scientific, Basingstoke, United Kingdom) uses a methodology similar to that of Etest. In this first assessment of the M.I.C. Evaluator device, 409 strains of aerobic Gram-positive bacteria (staphylococci, streptococci, and enterococci) and 325 strains of Enterobacteriaceae, Pseudomonas species, and Acinetobacter species were tested by M.I.C. Evaluator strip, Etest, and broth microdilution as a reference standard. The Gram-positive bacteria included staphylococci (methicillin-resistant Staphylococcus aureus, methicillin-susceptible S. aureus, and coagulase-negative staphylococci), Streptococcus pneumoniae, beta-hemolytic streptococci and viridians group strains, vancomycin-resistant enterococci, and other enterococci. The Gram-negative bacteria included 250 strains of 60 Enterobacteriaceae species plus 50 Pseudomonas and 25 Acinetobacter species. A total of 14 antimicrobial agents (depending on the species) were included. The same methodology and reading format were used for M.I.C. Evaluator strips and Etest. Broth microdilution methodology was performed according to CLSI document M07-A8. For the clinical strains, >95% of results were plus or minus one doubling dilution for all species. There were fewer than 5% minor errors, fewer than 3% major errors, and fewer than 1% very major errors. M.I.C. Evaluator strips and Etest often reported higher MICs than the reference broth microdilution method. The M.I.C. Evaluator strips provided results comparable to those of the predicate Etest device and are of value for the accurate testing of MICs for these important pathogens.

Interlaboratory study of quality control isolates for a broth microdilution method (modified CLSI M38-A) for testing susceptibilities of dermatophytes to antifungals.

The Clinical and Laboratory Standards Institute (CLSI; formerly National Committee for Clinical Laboratory Standards, or NCCLS) M38-A standard for the susceptibility testing of filamentous fungi does not specifically address the testing of dermatophytes. In 2003, a multicenter study investigated the reproducibility of the microdilution method developed at the Center for Medical Mycology, Cleveland, Ohio, for testing the susceptibility of dermatophytes. Data from that study supported the introduction of this method for testing dermatophytes in the future version of the CLSI M38-A standard. In order for the method to be accepted by CLSI, appropriate quality control isolates needed to be identified. To that end, an interlaboratory study, involving the original six laboratories plus two additional sites, was conducted to evaluate potential candidates for quality control isolates. These candidate strains included five Trichophyton rubrum strains known to have elevated MICs to terbinafine and five Trichophyton mentagrophytes strains. Antifungal agents tested included ciclopirox, fluconazole, griseofulvin, itraconazole, posaconazole, terbinafine, and voriconazole. Based on the data generated, two quality control isolates, one T. rubrum isolate and one T. mentagrophytes isolate, were identified and submitted to the American Type Culture Collection (ATCC) for inclusion as reference strains. Ranges encompassing 95.2 to 97.9% of all data points for all seven drugs were established.

Routine antimicrobial susceptibility testing of coagulase-negative staphylococci isolated from blood cultures: is it necessary?

The clinical significance of discontinuing routine antibiotic susceptibility testing (AST) of coagulase-negative Staphylococcus (CNS) isolates from blood cultures was investigated. Prospectively, AST was requested primarily for patients with serious underlying illnesses. Antibiotic use did not change significantly when AST was not performed routinely. Laboratory cost savings were 75% if AST was not performed, but more specimens were submitted from these patients. Oxacillin resistance in coagulase-negative staphylococci from blood cultures has remained > 70% since implementation of this protocol, while annual vancomycin utilisation has shown only small, incremental increases. Therefore, it is suggested that routine AST of CNS isolates from blood culture is not essential.

Development of a standardized susceptibility test for campylobacter with quality-control ranges for ciprofloxacin, doxycycline, erythromycin, gentamicin, and meropenem.

A standardized agar dilution susceptibility testing method was developed for Campylobacter that consisted of testing on Mueller-Hinton medium supplemented with 5% defibrinated sheep blood in an atmosphere of 10% CO2, 5% O2, and 85% N2. Campylobacter jejuni ATCC 33560 was identified as a quality-control (QC) strain. Minimal inhibitory concentration (MIC) QC ranges were determined for two incubation time/temperature combinations: 36 degrees C for 48 hr and 42 degrees C for 24 hr. Quality-control ranges were determined for ciprofloxacin, doxycycline, erythromycin, gentamicin, and meropenem. For all antimicrobial agents tested at both temperatures, 95-100% of the QC MIC results fell within recommended QC ranges. Twenty-one Campylobacter clinical isolates, encompassing five species of Campylobacter (C. jejuni, C. coli, C. jejuni, subsp. doylei, C. fetus, and C. lari) were tested in conjunction with the C. jejuni QC strain. While C. jejuni and C. coli could be reliably tested under both test conditions, growth of C. jejuni subsp. doylei, C. fetus, and C. lari isolates was inconsistent when incubated at 42 degrees C. Therefore, it is recommended that these species only be tested at 36 degrees C.

Quality control limits for voriconazole disk susceptibility tests on Mueller-Hinton agar with glucose and methylene blue.

An international collaborative study was performed in order to propose quality control limits for voriconazole disk diffusion tests on Mueller-Hinton agar with 2% glucose and 0.5 micro g of methylene blue per ml. The supplement may be added to the agar before autoclaving, or Mueller-Hinton agar plates may be flooded with a glucose-methylene blue solution. Replicate tests on both types of agar plates with 1- micro g voriconazole disks generated data to propose zone size limits for tests of Candida parapsilosis ATCC 22019 (28 to 37 mm), Candida albicans ATCC 90028 (31 to 42 mm), and Candida krusei ATCC 6258 (16 to 25 mm). Candida tropicalis ATCC 750 was not useful for this purpose.

Quality control limits for fluconazole disk susceptibility tests on Mueller-Hinton agar with glucose and methylene blue.

An international collaborative study was performed in order to propose quality control limits for fluconazole disk diffusion susceptibility tests on Mueller-Hinton agar with 2% glucose and 0.5 micro g of methylene blue per ml. The supplements may be added before autoclaving the agar, or Mueller-Hinton agar plates may be flooded with a glucose-methylene blue solution. Replicate tests on both types of agar plates generated data to propose zone size limits for tests of Candida parapsilosis ATCC 22019 (22 to 33 mm), C. tropicalis ATCC 750 (26 to 37 mm), and C. albicans ATCC 90028 (28 to 39 mm). C. krusei ATCC 6258 was not useful for this purpose.

Precision and accuracy of fluconazole susceptibility testing by broth microdilution, Etest, and disk diffusion methods.

Interpretive agreements among the results of fluconazole broth microdilution tests, Etests, and disk diffusion tests were documented by evaluating 495 Candida spp. Microdilution reference test results were in agreement with 96% of the Etest results; most discrepancies were minor differences. Fluconazole resistance of Candida krusei strains often required a full 48 h of incubation in order to be observed by the standard method. For the disk diffusion tests that were performed on Mueller-Hinton agar with glucose and methylene blue, 97% of results were in agreement with those of the reference test, especially when zones of inhibition were measured after the first 24 h of incubation. Some Candida glabrata isolates failed to grow satisfactorily until a full 48 h of incubation was completed. Precision was determined by testing 50 selected isolates in triplicate in each of three laboratories. The reproducibility of results of disk diffusion tests was comparable to that of the reference method. With all procedures, determination of test results was particularly challenging with some strains, and new methods are needed in order to improve endpoint definition.

Sputum isolation of Wangiella dermatitidis in patients with cystic fibrosis.

We report a case of invasive fungal pulmonary infection in a cystic fibrosis patient. Clinical deterioration coincided with isolation of Wangiella dermatitidis from her sputum, and treatment with amphotericin B followed by voriconazole resulted in clinical improvement and sterilization of the sputum. This case suggests that W. dermatitidis may be an etiologic agent of invasive pulmonary disease in the cystic fibrosis population.

Detection of anthrax spores in endemic regions of northern Canada.

AIMS: To determine the level of anthrax spore contamination in endemic regions of northern Canada between outbreaks. METHODS AND RESULTS: Bacterial endospores were extracted from specimens via flotation and cultured on selective PLET medium. Of 588 environmental specimens collected, 11 (1.9%) contained viable anthrax spores. CONCLUSION: High environmental concentrations of anthrax spores in northern Canada appear limited to scavenger faeces and anthrax carcass sites. Burial and cremation appear equally effective at removing anthrax spores from the immediate environment, though cremation may be improved by re-burning cremation sites containing unburned animal hair. SIGNIFICANCE AND IMPACT OF THE STUDY: This study describes an effective anthrax spore detection system. It provides the first bacteriological evidence that mammalian scavengers can disseminate anthrax spores in northern Canada, and its results may be compared with future environmental studies of untreated anthrax carcass sites to help improve government response plans.

Evaluation of E test, disk diffusion and broth microdilution to establish tentative quality control limits and review susceptibility breakpoints for two aerobic actinomycetes.

A single laboratory study was carried out to compare E Test with broth microdilution and disk diffusion to establish tentative quality control ranges for Nocardia asteroides ATCC 19247 and Rhodococcus equi ATCC 6939 against a panel of eight antimicrobial agents. Reproducibility testing was performed on 12 consecutive days to establish tentative quality control ranges. A total of 36 clinical strains of the Nocardia asteroides complex and 5 Rhodococcus strains were used in the study. Both candidate control strains and clinical strains grew well on cation-adjusted Mueller-Hinton agar. Adequate growth occurred at 48 to 72 h for the Nocardia isolates and 24 to 48 h for Rhodococcus. A standardized primary inoculum of 5 x 10(4) CFU/mL was used for performance of E Test and disk diffusion for the Nocardia isolates. Tentative population-based error rates were calculated using current breakpoints for Enterobacteriaceae for E Test compared with disk diffusion for the 36 clinical strains of Nocardia species. Significant very major error rates were observed for imipenem (22%) and minor error rates varied from 2.7% to 50%. These methods require more extensive validation before definitive breakpoint criteria can be established.

Evaluation of spore extraction and purification methods for selective recovery of viable Bacillus anthracis spores.

AIMS: To investigate methods of improving anthrax spore detection with PLET. METHODS AND RESULTS: Comparisons were made of PLET and blood-supplemented PLET to recover and distinguish spores of a variety of Bacillus species. Heat and ethanol purification of spores, and spore extraction from soil with water and high specific gravity sucrose plus non-ionic detergent, were also carried out. CONCLUSION: PLET was more selective and suitable than blood-supplemented PLET for detection of anthrax spores in the environmental specimens. However, PLET is not an optimal spore recovery medium. Purification of spores with ethanol was as effective as heat purification. High specific gravity sucrose plus detergent extraction solutions may be more sensitive than extraction with water. SIGNIFICANCE AND IMPACT OF THE STUDY: This study highlights shortcomings with the standard PLET isolation of anthrax spores and describes ways in which the procedure may be improved.

Novel fluorescent broth microdilution method for fluconazole susceptibility testing of Candida albicans.

A comparative evaluation of the reference National Committee for Clinical Laboratory Standards (NCCLS) broth microdilution method with a novel fluorescent carboxyfluorescein diacetate (CFDA)-modified microdilution method for the susceptibility testing of fluconazole was conducted with 68 Candida strains, including 53 Candida albicans, 5 Candida tropicalis, 5 Candida glabrata, and 5 Candida parapsilosis strains. We found trailing endpoints and discordant fluconazole MICs of < 8 microg/ml at 24 h and of > or =64 microg/ml at 48 h for 12 of the C. albicans strains. These strains satisfy the definition of the low-high MIC phenotype. All 12 low-high phenotype strains were correctly shown to be susceptible at 48 h with the CFDA-modified microdilution method. For the 41 non-low-high phenotype C. albicans strains, the CFDA-modified microdilution method yielded 97.6% (40 of 41 strains) agreement within +/-1 dilution at 24 h compared with the reference method and 92.7% (38 of 41 strains) agreement within +/-1 dilution at 48 h compared with the reference method. The five strains each from C. tropicalis, C. glabrata, and C. parapsilosis that were tested showed 100% agreement within +/-2 dilutions for the two methods being evaluated.

Characterization of Pseudomonas aeruginosa isolates: occurrence rates, antimicrobial susceptibility patterns, and molecular typing in the global SENTRY Antimicrobial Surveillance Program, 1997-1999.

During 1997-1999, a total of 70,067 isolates (6631 Pseudomonas aeruginosa isolates) were analyzed in the SENTRY program by geographic region and body site of infection. The respiratory tract was the most common source of P. aeruginosa. P. aeruginosa isolation rates increased during the study interval. Europe was the only region to show a significant decline in beta-lactam and aminoglycoside susceptibility rates. There was a reduction in the rates of susceptibility of Canadian isolates to imipenem and of Latin American isolates to meropenem. A total of 218 multidrug-resistant P. aeruginosa isolates (MDR-PSA; resistant to piperacillin, ceftazidime, imipenem, and gentamicin) were observed; MDR-PSA occurrence rates (percentages of all isolates) ranged from 8.2% (Latin America) to 0.9% (Canada). No antimicrobial inhibited >50% of MDR-PSA strains. Molecular characterization of selected, generally resistant strains was performed. Isolates showing unique ribogroups were found in Europe, Latin America, and the United States, but clonal spread was documented in several medical centers.

Quality control limits for broth microdilution susceptibility tests of ten antifungal agents.

Broth microdilution susceptibility tests of Candida species have now been standardized by the National Committee for Clinical Laboratory Standards (NCCLS). An eight-laboratory collaborative study was carried out in order to document reproducibility of tests of Candida parapsilosis ATCC 22019 and Candida krusei ATCC 6258 by the NCCLS method. Replicate broth microdilution tests were used to define control limits for 24- and 48-h MICs of amphotericin B, flucytosine, fluconazole, voriconazole, ketoconazole, itraconazole, caspofungin (MK 0991), ravuconazole (BMS 207147), posaconazole (SCH 56592), and LY 303366.