Invasive fungal infections by opportunistic yeasts have increased concomitantly with the growth of an immunocompromised patient population. Misidentification of yeasts can lead to inappropriate antifungal treatment and complications. Attenuated total reflectance Fourier transform infrared (ATR-FTIR) spectroscopy is a promising method for rapid and accurate identification of microorganisms. ATR-FTIR spectroscopy is a standalone, inexpensive, reagent-free technique that provides results within minutes after initial culture. In this study, a comprehensive spectral reference database of 65 clinically relevant yeast species was constructed and tested prospectively on spectra recorded (from colonies taken from culture plates) for 318 routine yeasts isolated from various body fluids and specimens received from 38 microbiology laboratories over a 4-month period in our clinical laboratory. ATR-FTIR spectroscopy attained comparable identification performance with matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS). In a preliminary validation of the ATR-FTIR method, correct identification rates of 100% and 95.6% at the genus and species levels, respectively, were achieved, with 3.5% unidentified and 0.9% misidentified. By expanding the number of spectra in the spectral reference database for species for which isolates could not be identified or had been misidentified, we were able to improve identification at the species level to 99.7%. Thus, ATR-FTIR spectroscopy provides a new standalone method that can rival MALDI-TOF MS for the accurate identification of a broad range of medically important yeasts. The simplicity of the ATR-FTIR spectroscopy workflow favors its use in clinical laboratories for timely and low-cost identification of life-threatening yeast strains for appropriate treatment.
Background Staphylococcus aureus is well known to be associated with atopic dermatitis. Recent studies also report S. aureus presence in lesional skin of squamous cell carcinoma (SCC) and its precursor lesion, actinic keratosis (AK). Therefore, it is of potential clinical interest to monitor skin S. aureus colonization on AK lesions. Fourier transform infrared (FTIR) spectroscopy is a cost-effective, nondestructive, and reagent-free technique for rapid microbial identification. It is based on the use of spectral databases developed with well-characterized strains in conjunction with the application of multivariate statistical analysis to elaborate classification models. In the present cross-lab study, spectral databases containing FTIR spectra of over 1000 staphylococcal isolates obtained from reference and clinical microbiology laboratories across Canada were employed in the FTIR spectroscopic identification of Staphylococcus spp. isolated from AK, SCC and perilesional skin of patients at the Princess Alexandra Hospital Dermatology Clinic in Brisbane, Australia.MethodsFTIR spectra of 51 staphylococcal isolates from AK, SCC and perilesional skin were acquired by both attenuated total reflectance (ATR)-FTIR and transflection-FTIR spectroscopy. All isolates had been previously characterized by 16S rRNA sequencing. ATR- and transflection-FTIR spectra were recorded in triplicate from isolated colonies taken from the same agar plate. Identification of the bacteria was based on the similarities of their spectra with those in ATR- and transflection-FTIR spectral databases originating from the Canadian lab.ResultsAmong the 51 staphylococcal isolates included in this study, identification of S. aureus (n = 24) with 100% specificity and 100% sensitivity was achieved by both ATR- and transflection-FTIR spectroscopy. Overall, FTIR-based species identification was in 90.2% concordance with 16S rRNA sequencing.ConclusionThis cross-lab study demonstrates the applicability of Canadian isolate-based ATR- and transflection-FTIR spectral databases for the identification of clinical staphylococcal isolates obtained in Australia. The results support the potential utility of FTIR spectroscopic techniques to monitor skin S. aureus colonization on AK lesions.Disclosures All authors: No reported disclosures.
Background Burkholderia cepacia complex including B. gladioli are opportunistic pathogenic bacteria affecting the immunocompromised population. For prognosis and appropriate treatment, rapid and accurate species identification is particularly important for those diagnosed with cystic fibrosis (CF). Conventional biochemical identification techniques are insensitive and problematic for identifying Burkholderia spp., leading to common misidentification or inconclusive results. Recent studies have successfully employed attenuated total reflectance-Fourier transform infrared (ATR-FTIR) spectroscopy for rapid, reagent-free and cost-effective microbial identification. In the present study, identification of Burkholderia spp. by this technique is investigated.MethodsA total of 59 isolates belonging to 7 species of Burkholderia were included in this study; all these isolates had been well-characterized by VITEK 2, 16S rRNA sequencing, random amplification of polymorphic DNA (recA typing) and/or matrix-assisted laser desorption/ionization time of flight mass spectrometry. ATR-FTIR spectra were acquired directly from colonies on 5% blood agar plates.ResultsA spectral database containing ATR-FTIR spectra of over 4300 bacterial isolates, encompassing over 70 genera and 190 species, was updated to include spectra of 39 isolates collected in this study and employed in the identification of the other isolates (n = 20). All isolates were correctly identified as Burkholderia by a multitier search approach. For Burkholderia species identification, spectra belonging to 39 isolates representative of all 7 species were used to construct a spectral database employed to identify the other 20 isolates [B. anthina (n = 2), B. gladioli (n = 8), B. multivorans (n = 7), and B. vietnamiensis (n = 3)]. Compared with VITEK 2 (30% correct species identification), ATR-FTIR spectroscopy correctly identified all but one isolate, resulting in overall correct species identification of 95%. Prospectively (10 months), 5 of 1100 isolates collected were identified as Burkholderia spp. by ATR-FTIR spectroscopy in concordance with VITEK 2.ConclusionATR-FTIR spectroscopy can provide the means of rapid Burkholderia spp. identification for appropriate treatment of those diagnosed with CF.Disclosures All authors: No reported disclosures.
BackgroundChronic respiratory infections with non-fermenting Gram-negative bacilli are a key feature of cystic fibrosis (CF). For microbiology laboratories, rapid and accurate identification of these bacteria is often challenging and labor intensive. This study was undertaken to evaluate whether attenuated total reflectance Fourier transform infrared (ATR-FTIR) spectroscopy could rapidly discriminate Pseudomonas aeruginosa (mucoid and non-mucoid), Burkholderia cepacia complex, Burkholderia gladioli, Achromobacter spp. and Stenotrophomonas maltophilia.MethodsA total of 263 well-characterized clinical strains isolated from respiratory samples of patients with CF attending the CHU Sainte-Justine CF clinic were included in this study, consisting of 70 P. aeruginosa, 83 Burkholderia spp., 52 Achromobacter spp. and 58 Stenotrophomonas maltophilia isolates from the biobank. Isolates were thawed and sub-cultured twice on sheep blood (5%) agar. ATR-FTIR spectral acquisition was performed in triplicate for each isolate. Multivariate statistical analysis of the ATR-FTIR spectra was performed by hierarchical cluster analysis (HCA) and principal component analysis (PCA) in conjunction with the use of a feature selection algorithm.ResultsAn ATR-FTIR spectral database consisting of 789 spectra of P. aeruginosa, Burkholderia spp., Achromobacter spp. and Stenotrophomonas maltophilia was created in this study. Complete discrimination among all four genera as well as among three species within the B. cepacia complex and B. gladioli was achieved based on HCA and PCA of the spectra in the database. ATR-FTIR analysis of a validation set consisting of 30 isolates was conducted in parallel with identification by MALDI-TOF mass spectrometry and yielded >95% concordance between the two techniques.ConclusionATR-FTIR spectroscopy is a promising tool for rapid, inexpensive and accurate identification of non-fermenting Gram-negative bacilli. Additional work is needed to further expand the spectral database, particularly with mucoid strains.Disclosures All authors: No reported disclosures.
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