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 manufacturer's 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. C urrently, there are more than 170 recognized species and subspecies of mycobacteria, more than 100 Nocardia species, and several hundred other aerobic actinomycetes species distributed across approximately 16 genera (1). Some of these organisms are clinically relevant and cause a spectrum of disease presentations in humans, while others are environmental organisms that can be found as commensal organisms or in laboratory cultures as contaminants resulting from specimen collection or processing (2). Accurate identification of mycobacteria and the aerobic actinomycetes is important for patient care but can be difficult due to the low growth rates of some species, the large number of species which have small differences in genetic diversity, and the need for biosafety level (BSL) 3 facilities when unknown isolates that might be Mycobacterium tuberculosis or Mycobacterium bovis are processed. The current gold standard for the identification of mycobacteria and aerobic actinomycetes is DNA sequencing with several targets recognized as useful for the species identification of mycobacteria and aerobic actinomycetes, including the 16S rRNA gene, rpoB, secA, and hsp65 (3). However, many clinical laboratories do not have the resources to routinely perform sequencing because it is labor-intensive and technically complex.In the last few years, matrix-assisted laser desorption ionizationϪtime of flight mass spectrometry (MALDI-TOF MS) has proven to be a reliable method for the identification of a wide variety of bacteria and yeasts following growth on culture medium (4-8). Fewer studies have ...

Nocardia species are found worldwide and are opportunistic pathogens of both immunocompromised and immunocompetent hosts. Recent updates to the taxonomy of this genus have indicated that there are more than 90 recognized species of Nocardia with 54 species reported to be clinically relevant. In this paper, we report the species distribution, specimen source distribution, and antimicrobial susceptibility profiles of 2,091 clinical isolates recovered for the years 2011 to 2017 using the updated taxonomy. The most commonly isolated species included Nocardia nova complex, Nocardia cyriacigeorgica, and Nocardia farcinica complex, with an additional 25 species or species complexes recovered from clinical specimens. The antimicrobial susceptibility profile was highly variable between the species, but in general, amikacin, linezolid, and trimethoprim-sulfamethoxazole demonstrated good in vitro activity against most species.

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.Cryptococcus gattii (formerly Cryptococcus neoformans var. gattii) differs from the closely related C. neoformans in several ways, including a contrasting host profile and a reduced susceptibility to certain antifungal agents (19,20). Cryptococcus gattii has traditionally been thought to be geographically restricted to tropical and subtropical climates, although recent reports indicate that it can be found worldwide, including in regions with distinctly nontropical climates, like the Pacific Northwest (2,4,8,12,14,17,19). Clinically, the majority of cryptococcosis cases that occur in AIDS patients and other immunocompromised hosts are caused by C. neoformans var. neoformans or C. neoformans var. grubii. In contrast, C. gattii has been reported to cause meningoencephalitis and pulmonary infections in hosts who are generally immune competent (9,14,18).Since C. gattii is recognized as an emerging pathogen, it is important for the clinical microbiology laboratory to accurately differentiate it from C. neoformans. A recent proficiency testing survey administered by the New York State Department of Health indicated that only 7/140 (5%) clinical laboratories participating in the event were able to correctly identify C. gattii, while the remaining 95% of laboratories surveyed misidentified the isolate as C. neoformans (15). The critique that followed this proficiency event in...

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. N ocardia species are ubiquitous in the environment and have been implicated in a variety of human infections, especially in immunocompromised patients. In these patients, infection most frequently begins in the lungs, but can quickly disseminate to nearly every organ of the body. Nocardia can also cause disease in the immunocompetent host, in whom it is usually introduced through traumatic injury (2).Nocardia species are known to vary in their susceptibilities to various antimicrobial agents, and some species, especially Nocardia farcinica and members of the Nocardia transvalensis complex, have been shown to be particularly resistant to commonly used antimicrobial agents. Therefore, susceptibility testing should be performed on any Nocardia isolate considered to be of possible clinical significance.In 2003, the Clinical and Laboratory Standards Institute (CLSI) published an approved standard (M24A) for susceptibility testing of mycobacteria, nocardiae, and other aerobic actinomycetes, which specifies the broth microdilution method as the recommended procedure for susceptibility testing of aerobic actinomycetes (6). This document has recently been updated (5). Several years of experience in testing Nocardia isolates at the University of Texas Health Science Center and at the National Institutes of Health have demonstrated difficulties that may occur in inoculum preparation due to the propensity for clumping demonstrated by these organisms. In addition, determination of susc...

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. Candida species are the leading cause of fungemia in both community-acquired and nosocomial infections (2). Two recent studies have shown that the use of PNA FISH probes to identify yeast in blood cultures decreases patient costs by directing appropriate antimicrobial therapy. Forrest et al. used the FDA-cleared Candida albicans PNA FISH kit (AdvanDx, Woburn, MA) for identification of C. albicans in 72 positive blood cultures (3). Rapid results allowed for a significant reduction in the use of caspofungin, with an overall cost savings of $1,729 per patient. In a similar study, Alexander and colleagues used a decision analytic model and showed an average savings of $1,837 per patient with the use of PNA FISH (1). Since the publication of these and other studies (6, 7, 9), additional probe kits have been cleared by the FDA, including the C. albicans/Candida glabrata PNA FISH kit and the Yeast Traffic Light PNA FISH kit (YTL).The performance of the C. albicans/C. glabrata PNA FISH kit has been previously reported (8). The YTL is a next-generation, three-probe system which is FDA cleared for the rapid identification of C. albicans/Candida parapsilosis, C. glabrata/Candida krusei and Candida tropicalis from positive blood cultures. Initially, the YTL was cleared using a 90-min probe hybridization step. Recently, a 30-min hybridization time was cleared, which further reduced the turnaround time to less than 2 h after the blood culture bottle signals as positive. In this study, we report the performance characteristics of the YTL kit using both the 90-and 30-min hybridization times. The reliability of the kit for the identification of the five claimed Candida species was evaluated, and the specificity of the probes was challenged using a wide variety of other yeast genera and species.Fifty-four blood cultures that were positive by Gram stain (44 yeast and 10 bacteria) were tested on the day of signal or were aliquoted and frozen at Ϫ20°C before testing as per the YTL product insert. In order to increase the number and diversity of yeasts tested, an additional 118 yeasts were inoculated into 5 ml of human red blood cells (Innovative Research, Navi, MI), and the simulated blood specimen was then placed into a Bactec MYCO/F lytic blood culture bottle (Becton Dickinson, Sparks, MD), incubated at 37°C, and tested with the YTL kit on the day that the bottle signaled positive. Subcultures of an additional 44 unusual yeast species grown on inhibitory mold agar (Becton Dickinson) were also tested for potential cross-reactivity of nontargeted yeast. The reference method for identification of the yeast used in the study was Sanger sequencing of the D2 large-subunit (LSU) RNA g...