Sharon M. Deml

Evaluation of the Yeast Traffic Light® PNA FISH™ Probes for Identification of Candida species from Positive Blood Cultures

ABSTRACT The Yeast Traffic Light PNA FISH kit (YTL) correctly identified Candida spp. in 207/216 (96%) positive blood cultures. Discordant results were seen with known cross-reacting species and cultures containing Candida lambica and Rhodotorula mucilaginosa. The YTL provides rapid, reliable identification of the five common Candida species found in blood cultures.

Identification of Cryptococcus gattii by Use of l-Canavanine Glycine Bromothymol Blue Medium and DNA Sequencing

ABSTRACT Cryptococcus neoformans and Cryptococcus gattii are closely related pathogenic fungi. Cryptococcus neoformans is ecologically widespread and affects primarily immunocompromised patients, while C. gattii is traditionally found in tropical climates and has been reported to cause disease in immunocompetent patients. l-Canavanine glycine bromothymol blue (CGB) agar can be used to differentiate C. neoformans and C. gattii, but there are few reports of its performance in routine clinical practice. Growth of C. gattii on CGB agar produces a blue color, indicating the assimilation of glycine, while C. neoformans fails to cause a color change. Using reference and clinical strains, we evaluated the ability of CGB agar and D2 large ribosomal subunit DNA sequencing (D2 LSU) to differentiate C. neoformans and C. gattii. One hundred two yeast isolates were screened for urease activity, melanin production, and glycine assimilation on CGB agar as well as by D2 sequencing. Seventeen of 17 (100%) C. gattii isolates were CGB positive, and 54 of 54 C. neoformans isolates were CGB negative. Several yeast isolates other than the C. gattii isolates were CGB agar positive, indicating that CGB agar cannot be used alone for identification of C. gattii. D2 correctly identified and differentiated all C. gattii and C. neoformans isolates. This study demonstrates that the use of CGB agar, in conjunction with urea hydrolysis and Niger seed agar, or D2 LSU sequencing can be reliably used in the clinical laboratory to distinguish C. gattii from C. neoformans. We describe how CGB agar and D2 sequencing have been incorporated into the yeast identification algorithm in our laboratory.

Evaluation of Matrix-Assisted Laser Desorption Ionization−Time of Flight Mass Spectrometry for Identification of Mycobacterium species, Nocardia species, and Other Aerobic Actinomycetes

ABSTRACT The value of matrix-assisted laser desorption ionization−time of flight mass spectrometry (MALDI-TOF MS) for the identification of bacteria and yeasts is well documented in the literature. Its utility for the identification of mycobacteria and Nocardia spp. has also been reported in a limited scope. In this work, we report the specificity of MALDI-TOF MS for the identification of 162 Mycobacterium species and subspecies, 53 Nocardia species, and 13 genera (totaling 43 species) of other aerobic actinomycetes using both the MALDI-TOF MS manufacturers supplied database(s) and a custom database generated in our laboratory. The performance of a simplified processing and extraction procedure was also evaluated, and, similar to the results in an earlier literature report, our viability studies confirmed the ability of this process to inactivate Mycobacterium tuberculosis prior to analysis. Following library construction and the specificity study, the performance of MALDI-TOF MS was directly compared with that of 16S rRNA gene sequencing for the evaluation of 297 mycobacteria isolates, 148 Nocardia species isolates, and 61 other aerobic actinomycetes isolates under routine clinical laboratory working conditions over a 6-month period. MALDI-TOF MS is a valuable tool for the identification of these groups of organisms. Limitations in the databases and in the ability of MALDI-TOF MS to rapidly identify slowly growing mycobacteria are discussed.

Multisite Reproducibility of the Broth Microdilution Method for Susceptibility Testing of Nocardia Species

ABSTRACT Antimicrobial susceptibility testing (AST) of clinical isolates of Nocardia is recommended to detect resistance to commonly used antimicrobial agents; such testing is complicated by difficulties in inoculum preparation and test interpretation. In this study, six laboratories performed repetitive broth microdilution testing on single strains of Nocardia brasiliensis, Nocardia cyriacigeorgica, Nocardia farcinica, Nocardia nova, and Nocardia wallacei. For each isolate, a total of 30 microdilution panels from three different lots were tested at most sites. The goal of the study was to determine the inter- and intralaboratory reproducibility of susceptibility testing of this group of isolates. Acceptable agreement (>90% agreement at ±1 dilution of the MIC mode) was found for amikacin, ciprofloxacin, clarithromycin, and moxifloxacin. After eliminating MIC values from single laboratories whose results showed the greatest deviation from those of the remaining laboratories, acceptable agreement was also found for amoxicillin-clavulanic acid, linezolid, minocycline, and tobramycin. Results showed unsatisfactory reproducibility of broth microdilution testing of ceftriaxone with N. cyriacigeorgica and N. wallacei, tigecycline with N. brasiliensis and N. cyriacigeorgica, and sulfonamides with N. farcinica and N. wallacei. N. nova ATCC BAA-2227 is proposed as a quality control organism for AST of Nocardia sp., and the use of a disk diffusion test for sulfisoxazole is proposed as a check of the adequacy of the inoculum and to confirm sulfonamide MIC results.

Evaluation of Mycobacterium avium Complex Clarithromycin Susceptibility Testing Using SLOMYCO Sensititre Panels and JustOne Strips

ABSTRACT The SLOMYCO Sensititre panel and the custom JustOne strip (both from TREK Diagnostic Systems, Cleveland, OH) were evaluated for susceptibility testing of Mycobacterium avium complex isolates against clarithromycin. Seventy-one archived and prospectively collected isolates were tested using both the SLOMYCO panel and the JustOne strip, and the results were compared to those obtained using the BACTEC 460 (BD, Sparks, MD) radiometric method and a broth microdilution reference method. Results obtained by the SLOMYCO panel and the JustOne strip agreed with the BACTEC 460 method for 64/71 isolates (90%). Similarly, concordance with the broth microdilution method was 40/43 isolates (93%) for both test systems. The effect of the source medium on inoculum preparation was evaluated, and there were no differences noted in MICs, regardless of whether the inoculum was prepared from isolates grown in Middlebrook 7H9 medium, on Middlebrook 7H10 agar, or in VersaTREK broth culture bottles (Trek Diagnostics). Clarithromycin susceptibility testing of MAC using the SLOMYCO panel and the JustOne strip methods is easy to set up and simple to read and is readily incorporate into the clinical laboratory. These systems offer advantages over the BACTEC 460 system including the lack of a need for radioactive substrates, sharps, or costly instrumentation.

Mycobacterium arupense Flexor Tenosynovitis: Case Report and Review of Antimicrobial Susceptibility Profiles for 40 Clinical Isolates

ABSTRACT We describe a case of chronic tenosynovitis in the hand of a 58-year-old cattle farmer. Surgical biopsy specimens grew Mycobacterium arupense. The patient responded to surgery and antimicrobial therapy based on in vitro susceptibility testing. The antimicrobial susceptibility profiles of the isolate from this patient and 39 additional clinical isolates are presented.