Practical Utility and Accuracy of Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry for Identification of Corynebacterium Species and Other Medically Relevant Coryneform-Like Bacteria.

BACKGROUND: Corynebacterium species and gram-positive coryneform-like bacteria (coryneforms) are increasingly reported causes of opportunistic infections in immunocompromised patients. Biochemical identification methods for these bacteria are often inaccurate. We evaluated matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) for routine Corynebacterium and coryneform identification. METHODS: A total of 286 Corynebacterium species and coryneforms recovered from patients were identified by MALDI-TOF MS analysis using the Bruker Microflex instrument, Biotyper software version 3.0, and database version 3.1.66 (Bruker Daltonics, Billerica, MA) following formic acid-based, direct on-plate extraction. The spectral cutoff scores used for interpretation were 2.0 or more for species-level identification and 1.7 or more for genus level. Scores lower than 1.7 were considered as no reliable identification. The results were compared with API Coryne (bioMérieux, Durham, NC) and sequencing of 16S ribosomal RNA genes. RESULTS: Of the 231 Corynebacterium (19 species), 99.6% were correctly identified to the genus level and 88.7% to the species level. Of the 55 coryneforms (14 genera), 90.9% were correctly identified to the genus level and 67.3% to the species level. API Coryne was able to identify 89.2% of Corynebacterium species (species level) and 63.6% of coryneforms (genus level). CONCLUSIONS: Rapid on-plate testing yielded identification of more Corynebacterium species and related bacteria than biochemical methods.

Fusarium solani species complex associated with carapace lesions and branchitis in captive American horseshoe crabs Limulus polyphemus.

Captive American horseshoe crabs Limulus polyphemus housed at the National Aquarium presented with a variety of shell and gill lesions over a 3 yr period. Carapace lesions were located on both the dorsal and ventral prosoma and opisthosoma and included multifocal circular areas of tan discoloration, ulcerations, and/or pitting lesions, extending from superficial to full thickness. Gill lesions involved both the book gill cover (operculum) and individual book gill leaflets and included multifocal circular areas of tan discoloration, tan to off-white opaque proliferative lesions, and/or areas of black discoloration. Histopathology revealed fungal hyphae, with variable morphology throughout the thickened and irregular cuticle of the carapace and occasionally penetrating into subcuticular tissues, with associated amebocytic inflammation. Book gill leaflets were infiltrated by fungal hyphae and contained necrotic debris and amebocytes. Thirty-eight of 39 animals (97%) evaluated via histopathological examination had intralesional fungal hyphae. Fungal cultures of carapace and gill lesions were attempted in 26 tissue samples from 15 individuals and were positive in 13 samples (50%), with 10 cultures (77%) yielding identification to genus. Fusarium sp. was identified in 8 of the 10 cultures (80%) via culture morphology. The Fusarium solani species complex was confirmed in 6 of these 8 (75%) via polymerase chain reaction amplification of 2 different ribosomal-specific sequences of isolated fungal DNA. Ante-mortem systemic and topical treatments were performed on some affected individuals, but no appreciable change in lesions was observed. Mycotic dermatitis and branchitis are serious health issues for captive American horseshoe crabs.

Evaluation of nucleic acid sequencing of the D1/D2 region of the large subunit of the 28S rDNA and the internal transcribed spacer region using SmartGene IDNS [corrected] software for identification of filamentous fungi in a clinical laboratory.

Filamentous fungal infections have recently increased because of the increasing numbers of immunocompromised hosts. In this study, we evaluated DNA sequencing of the D1/D2 region of the large subunit of the 28S ribosomal RNA gene and the internal transcribed spacer (ITS) region using SmartGene (SG; SmartGene Inc., Raleigh, NC) for the identification of a broad range of commonly encountered filamentous fungi. The SG proofreaders were used to upload, align, and edit fragments, and the resultant sequences were interpreted using the quality-controlled SG database. The results were compared with reference identifications using conventional phenotypic methods or ITS DNA sequences obtained from GenBank if phenotypic identifications were inconclusive. A total of 146 clinical isolates were included in this study, representing 49 different genera. The overall agreements of the D1/D2 and the ITS sequencing methods to reference identification were 97.2% (95% CI, 93.1% to 98.9%) and 97.7% (95% CI, 92.8% to 99.4%), respectively. Of the 146 isolates, 18 (12.3%) did not amplify using the ITS universal primers after repeated attempts and, therefore, could not be sequenced using this target. Correct identification was achieved for 100% (95% CI, 97.4% to 100%) of the isolates when applying both the D1/D2 and ITS targets. In summary, DNA sequencing using SG software provides a rapid, accurate, and reliable tool for the identification of filamentous fungi in a clinical laboratory.

Actinomucor elegans as an emerging cause of Mucormycosis.

We report an invasive mucormycosis caused by Actinomucor elegans in a patient with refractory aplastic anemia. The organism was isolated from a necrotic skin lesion on the patient's left arm and demonstrated angioinvasive features on histopathology examination. In contrast to three cases described previously, we describe the first case of A. elegans invasive fungal infection in an immunocompromised patient. This report, along with the three previously reported cases, is convincing evidence that A. elegans is an emerging fungal pathogen capable of causing invasive mucormycosis in humans.

Rapid, standardized method for determination of Mycobacterium tuberculosis drug susceptibility by use of mycolic acid analysis.

Multidrug-resistant (MDR) Mycobacterium tuberculosis and extrensively drug-resistant (XDR) M. tuberculosis are emerging public health threats whose threats are compounded by the fact that current techniques for testing the susceptibility of M. tuberculosis require several days to weeks to complete. We investigated the use of high-performance liquid chromatography (HPLC)-based quantitation of mycolic acids as a means of rapidly determining drug resistance and susceptibility in M. tuberculosis. Standard susceptibility testing and determination of the MICs of drug-susceptible (n = 26) and drug-resistant M. tuberculosis strains, including MDR M. tuberculosis strains (n = 34), were performed by using the Bactec radiometric growth system as the reference method. The HPLC-based susceptibilities of the current first-line drugs, isoniazid (INH), rifampin (RIF), ethambutol (EMB), and pyrazinamide (PZA), were determined. The vials were incubated for 72 h, and aliquots were removed for HPLC analysis by using the Sherlock mycobacterial identification system. HPLC quantitation of total mycolic acid peaks (TMAPs) was performed with treated and untreated cultures. At 72 h, the levels of agreement of the HPLC method with the reference method were 99.5% for INH, EMB, and PZA and 98.7% for RIF. The inter- and intra-assay reproducibilities varied by drug, with an average precision of 13.4%. In summary, quantitation of TMAPs is a rapid, sensitive, and accurate method for antibiotic susceptibility testing of all first-line drugs currently used against M. tuberculosis and offers the potential of providing susceptibility testing results within hours, rather than days or weeks, for clinical M. tuberculosis isolates.

Use of peptide nucleic acid-fluorescence in situ hybridization for definitive, rapid identification of five common Candida species.

We investigated a 2.5-h peptide nucleic acid-fluorescence in situ hybridization (PNA-FISH) assay with five Candida species-specific probes to identify Candida colonies and compared it to standard 2-h to 5-day phenotypic identification methods. Suspensions were made and slides were prepared and read for fluorescence per the manufacturer's instructions. Sensitivity was 99% (109/110), and specificity was 99% (129/130). PNA-FISH can rapidly identify those Candida species isolated most frequently.