FTIR spectroscopy in medical mycology: Applications to the differentiation and typing of Candida
Laboratoire de Parasitologie-Mycologie, CHU de Reims, EA 3800, IFR53, Université de Reims Champagne-Ardenne, 51 rue Cognacq-Jay, 51096 Reims Cedex, France. Analytical and Bioanalytical Chemistry
(Impact Factor: 3.44).
04/2007; 387(5):1729-37. DOI: 10.1007/s00216-006-1005-1
The incidence of fungal infections, in particular candidiasis and aspergillosis, has considerably increased during the last three decades. This is mainly due to advances in medical treatments and technologies. In high risk patients (e.g. in haematology or intensive care), the prognosis of invasive candidiasis is relatively poor. Therefore, a rapid and correct identification of the infectious agent is important for an efficient and prompt therapy. Most clinical laboratories rely on conventional identification methods that are based on morphological, physiological and nutritional characteristics. However, these have their limitations because they are time-consuming and not always very accurate. Moreover, molecular methods may be required to determine the genetic relationship between the infectious strains, for instance in Candida outbreaks. In addition, the latter methods require time, expensive consumables and highly trained staff to be performed adequately. In this study, we have applied the FTIR spectroscopic approach to different situations encountered in routine mycological diagnosis. We show the potentials of this phenotypic approach, used in parallel with routine identification methods, for the differentiation of 3 frequently encountered Candida species (C. albicans, C. glabrata and C. krusei) by using both suspensions and microcolonies. This approach, developed for an early discrimination, may help in the initial choice of antifungal treatment. Furthermore, we demonstrate the feasibility of the method for intraspecies comparison (typing) of 3 Candida species (C. albicans, C. glabrata and C. parapsilosis), particularly when an outbreak is suspected.
Available from: Mohamed Taha El Sayed
- "Despite the fact that conventional/classical tests are largely applied, yet, these tests are faced with several constrains. Time consuming and low discriminative power between closely related species are the major constrains . Currently, genotypic methods are practically preferred than phenotypic methods. "
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ABSTRACT: Rapid and accurate identification of yeast is increasingly important to stipulate the appropriate therapy thus reducing morbidity and mortality related to yeast infections. Vibrational spectroscopic techniques (infrared (IR) and Raman) could provide potential alternatives to conventional typing methods, because they constitute a rapid, inexpensive and highly specific spectroscopic fingerprint through-which microorganism can be identified. The present study evaluate (FTIR) spectroscopy as a sensitive and effective assay for the identification of the most frequent yeast species isolated from human and animals. One hundred and twenty-eight yeasts isolated from infected human mouths/vaginas, chronic diseased cows, crop mycosis in chicken and soil contaminated with pigeon droppings were phenotypically identified. Using universal primers, ITS1/ITS4, we have amplified ITS1-5.8S-ITS2 rDNA regions for 39 yeast isolates as representative samples. The PCR products were digested with restriction enzyme MspI and examined by PCR-RFLP, which was an efficient technique for identification of Candida spp., Cryptococcus neoformans and Trichosporon asahii. Further, identification of the same 39 isolates were done by FTIR spectroscopy and considered as reference for other strains by comparison of their FTIR spectra. The current study has sharply demonstrated the significant spectral differences between the various examined species of Candida, Cryptococcus, Trichosporon, Rhodotorula and Geotrichum isolated from different sources. Decisively, our research has confirmed that FTIR spectroscopy is a promising diagnostic tool, because of its sensitivity, rapidity, high differentiation capacity and simplicity compared to conventional/molecular techniques.
Available from: scielo.br
- "Eng., 28(4), 398-409, Dec. 2012 404 diferenciar os três espectros. Esse resultado demonstra que, mesmo nos casos em que os espectros de diferentes micro-organismos apresentem o mesmo aspecto visual, a análise estatística multivariada pode ser potencialmente útil para diferenciar os espectros desses micro-organismos (Essendoubi et al., 2005; Naumann, 2000; Timmins et al., 1998; Tintelnot et al., 2000; Toubas et al., 2007). "
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ABSTRACT: Films of Candida albicans, Candida dubliniensis and Candida parapsilosis were prepared and the infrared spectra of these films were obtained in the region 4000 to 1000 cm1, with resolution of 4 cm1, in the transmission mode, at 20 ºC. Fifty four spectra were obtained, 18 of each microorganism, with the PerkinElmer Spotlight 400 FT-IR, which has a microscope attached to a FT-IR spectrophotometer. The spectra were analyzed through three methods: (1) mere visual inspection; (2) multivariate statistical analysis; (3) curve-fitting for determining secondary structures of proteins. In the region 1200 to 1000 cm1, the spectral bands show differences that can be seen by a mere visual inspection. On the other hand, the amide I bands, in the region 1710 to 1590 cm1, have the same visual aspect for the three microorganisms. Multivariate statistical analysis was applied to analyze these amide I bands of all the 54 spectra. Principal component analysis (PCA) and techniques of hierarchical cluster analysis (HCA, Hierarchical Clustering Analysis) according to Ward's method were applied using the software MINITAB 15. The results show a clear discrimination of the three microorganisms. The average spectrum of each microorganism was obtained in the amide I band. Each average spectrum was analyzed by curve-fitting for the determination of secondary structures of proteins. The software used was the ORIGIN 7.5 and the results confirm the discrimination obtained through multivariate statistical analysis. This result shows that multivariate statistical analysis can be useful to discriminate infrared spectra of different microorganisms. Furthermore, this work shows that the amide I bands of the infrared spectra of Candida albicans, Candida dubliniensis, and Candida parapsilosis provide a set of data of known group structure that can be useful to test statistical algorithms of cluster analysis.
- "DRIFT offers among others the additional advantages of simpler sample preparation and the capacity to analyze nontransparent materials. FT-IR spectroscopy has been successfully applied in a wide range of studies, including identification/differentiation of various types of biological materials (Pappas et al. 2008; Tarantilis et al. 2008) as well as in the discrimination and metabolic responses of several bacteria and yeast species (Lamprell et al. 2006; Toubas et al. 2007; Kamnev 2008). As regards filamentous fungi in particular, FT-IR spectroscopy was used for examining a few groups of significant importance , e.g., causes of fungal infections in humans (Erukhimovitch et al. 2005), mycotoxins producing agents (Galvis-S anchez et al. 2008), and plant pathogens (Salman et al. 2010). "
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ABSTRACT: Fourier transform infrared (FT-IR) spectroscopy has been successfully applied for the identification of bacteria and yeasts, but only to a limited extent for discriminating specific groups of filamentous fungi. In the frame of this study, 73 strains - from different associated hosts/substrates and geographic regions - representing 16 taxa of the edible mushroom genus Pleurotus (Basidiomycota, Agaricales) were examined through the use of diffuse reflectance infrared Fourier transform (DRIFT) spectroscopy. A binary matrix, elaborated on the basis of presence/absence of specific absorbance peaks combined with cluster analysis, demonstrated that the spectral region 1800-600 cm(-1) permitted clear delimitation of individual strains into Pleurotus species. In addition, closely related species (e.g., Pleurotus ostreatus and Pleurotus pulmonarius) or taxa of the subgenus Coremiopleurotus demonstrated high similarity in their absorbance patterns, whereas genetically distinct entities such as Pleurotus dryinus, Pleurotus djamor, and Pleurotus eryngii provided spectra with noteworthy differences. When specific regions (1800-1700, 1360-1285, 1125-1068, and 950-650 cm(-1)) were evaluated in respect to the absorbance values demonstrated by individual strains, it was evidenced that this methodology could be eventually exploited for the identification of unknown Pleurotus specimens with a stepwise process and with the aid of a dichotomous key developed for this purpose. Moreover, it was shown that the nature of original fungal material examined (mycelium, basidiomata, and basidiospores) had an effect on the outcome of such analyses, and so did the use of different mycelium growth substrates. In conclusion, application of FT-IR spectroscopy provided a fast, reliable, and cost-efficient solution for the classification of pure cultures from closely related mushroom species.
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