Evaluation of Disseminated Candidiasis on an ExperimentalAnimal Model: A Fourier Transform Infrared Study

AYCA DOGAN, KIVANC ERGEN, FATMA BUDAK, and FERIDE SEVERCAN*Department of Biophysics (A.D., K.E.), Department of Microbiology and Clinical Microbiology (F.B.), Kocaeli University, Faculty of Medicine,

41380, Kocaeli-Turkey; and Department of Biology, Middle East Technical University, 06531, Ankara-Turkey (F.S.)

Invasive candidiasis, defined as candidemia and disseminated candidiasis,

is the most common fungal infection in hospitalized patients. In the

current study, we used Fourier transform infrared (FT-IR) spectroscopy

as a rapid, non-perturbing technique to investigate the effects of

disseminated candidiasis on mouse liver tissues at the molecular level.

The results revealed that the infection caused compositional changes in

the tissues by decreasing the lipid content and the ratio of the saturated

lipids to unsaturated lipids. An increase in the lipid/protein ratio was also

observed. In addition, investigation of the olefinic band at 3014 cm�1

showed that lipid peroxidation took place in the infected samples. These

results indicate that FT-IR spectroscopy is a promising technique for the

evaluation and diagnosis of disseminated candidiasis.

Index Headings: Fourier transform infrared spectroscopy; FT-IR spec-

troscopy; Infectious disease; Diagnosis; Candida albicans; Disseminated

candidiasis; Liver; Lipid; Protein.

INTRODUCTION

The fungus Candida albicans is one of the most commonfungal pathogens of humans. Candidiasis associated with C.albicans is primarily an opportunistic infection of immuno-compromised patients.1,2 In a previous study, organ involve-ment was most commonly identified in the lung with a rate of58%, followed by the liver (23%), kidney (16%), brain (12%),spleen (8%), and heart (8%).3 The most common presentingfeature of disseminated candidiasis is not diagnostic. Thepatient usually has fever, tachycardia, and dyspnea. Sometimesclinical symptoms cannot be differentiated from sepsis or septicshock caused by gram-negative bacteria. Culturing from liverbiopsies is also difficult. Disseminated candidiasis wasinvestigated from the aspect of molecular diagnosis,4–6

pathology of affected organs7 and genetics, pathogenicity,and immunology.8–11 Candidiasis-induced molecular changesin the tissues have not been yet reported in the literature.Furthermore, the use of analytical techniques for the diagnosisof this infection is very limited.12–14 In these studies, gaschromatography and mass spectroscopy, which were mainlyused in body fluids, and the magnetic resonance imagingtechnique were employed in liver and spleen biopsies.14–16 Thecurrent analytical methods do not address biochemical changes,which can provide early diagnosis of the diseases.

Fourier transform infrared (FT-IR) spectroscopy is avaluable analytical technique in biochemistry, biophysics,and molecular biology due to its high sensitivity in detectingchanges in the functional groups belonging to tissue compo-nents, such as lipids, proteins, and carbohydrates, simulta-neously. The shifts in peak positions and changes in thebandwidth, intensity, and more accurately, area values of the

bands, give valuable structural and functional information,which may have diagnostic value.17–19 FT-IR spectroscopytogether with microscopy has been widely used in themolecular investigation and diagnosis of many diseasesranging from diabetes and malignancy17,20–26 to infectiousdiseases.27,28

In the present study we investigated the effect ofdisseminated candidiasis on an animal experimental model.To achieve this, candidiasis-induced changes in mouse livertissues were determined at the molecular level by monitoringthe different vibrational functional groups. For the first time wedetermined the biochemical variations in between normal andinfectious samples. Furthermore, the diagnosis of this infec-tious disease has been achieved using cluster analysis based onFT-IR data, which to the best of our knowledge, has not beenreported previously.

MATERIALS AND METHODS

Animal Model and Inoculum, Infection, and Therapy. Atotal of 12 immunocompetent, healthy male BALB/c mice(age; six to eight weeks, weight; 25–30 g) were used in thestudy. They were housed in cages of one mouse each and hadaccess to food and water ad libutum. Mice were divided in totwo groups as control (n ¼ 6) and infected mice (n ¼ 6). Thecontrol group was uninfected and untreated, while the othergroups were infected with Candida albicans. The livers of themice were removed and stored at �80 8C for FT-IRspectroscopic analysis. All animal studies were approved bythe research ethical committee of Kocaeli University, MedicalFaculty, Turkey (No: 1231/2003).

Standard C. albicans isolate (ATCC 90028) was used in thisstudy. To prepare the infectious inoculum, C. albicans wasincubated overnight in Sabouraud 2% dextrose agar (SDA)(Merck, Germany). Yeast cells were washed three times andsuspended in normal sterile saline. This suspension was thenfurther diluted with saline until an optical density thatcorresponds to an inoculum of 107 colony-forming units(CFU) of C. albicans per milliliter was obtained, as determinedby quantitative plating in 1:10 dilution steps on SDA. Anamount of 0.2 mL of this suspension, which corresponds to aninoculum of 2 3 106 CFU of C. albicans, was administeredintravenously to each mouse through the lateral tail vein.29

Histologic Examination. Formalin-fixed liver tissue sam-ples were embedded in paraffin by routine methods. Serial liversections were obtained (5 lm in thickness) and were stainedwith Haematoxylin and Eosin and Mason Tricrom. Sectionswere examined by light microscopy (BX5OF; Olympus).

Fourier Transform Infrared Spectroscopic Analysis. Thesamples were ground in liquid nitrogen and dried overnight inLABCONCO lyo freeze drier. The samples were then groundwith extensively dried potassium bromide at a 1/100 ratio. This

Received 26 June 2006; accepted 4 December 2006.* Author to whom correspondence should be sent. E-mail: feride@metu.edu.tr.

Volume 61, Number 2, 2007 APPLIED SPECTROSCOPY 1990003-7028/07/6102-0199$2.00/0

� 2007 Society for Applied Spectroscopy

powder was compressed into a thin KBr disk under a pressureof approximately 1100 kg/ cm2 for 5 minutes.

Infrared spectra were obtained using a Perkin Elmer, FT-IR(Spectrum One) spectrometer equipped with a DTGS (deuter-ated triglycine sulfate) detector. Water vapor and carbondioxide interference were automatically subtracted. The FT-IRspectra of the samples were recorded in the 4000–450 cm�1

region at room temperature. One hundred (100) scans weretaken for each interferogram at 4 cm�1 spectral resolution.Three different pellets, prepared from the same sample, werescanned and identical spectra were obtained. These wereaveraged for each sample using the Perkin Elmer softwareprogram, for further comparison. All the other spectral analysesincluding baseline correction, normalization, and deconvolu-tion were performed using the same software program. Theband positions were measured from the center of weight of thepeaks. The spectra were normalized with respect to a specificband (amide A) for only the visual demonstration of thechanges so that the peak absorbance of the specific band is setto unity. However, for the precise determination of thevariations (band area and peak position), each of the originalbaseline-corrected spectra was used.

Cluster Analysis. Cluster analysis examines the interpointdistances between all the samples and represents the informa-

tion in the form of a two-dimensional plot, known as adendrogram. In a dendrogram, the cluster of samples wasformed based on their nearness in row space. For the spectraldifferentiation of experimental and control groups, clusteranalysis was applied by using OPUS 5.5 software. Vectornormalization was performed in the two different spectralregions, namely 4000–450 cm�1 and 1950–450 cm�1. As inputdata for cluster analysis, spectral distances were calculatedbetween pairs of spectra as Pearson’s correlation coefficients.Cluster analysis for the separation of control and infectedtissues were based on the Euclidean distances. In all cases,Ward’s algorithm was used for hierarchical clustering.

Statistical Analysis. The Mann–Whitney U-test wasperformed on the groups to test the significance of thedifferences between the control and infected groups. A p value,0.05 was considered to be statistically significant.

RESULTS AND DISCUSSION

We carried out FT-IR spectroscopic studies of normal anddisseminated candidiasis mouse liver tissues to identify thebiological changes occurring in the system at the molecularlevel. Histologically, it was proven that the mice were infected.In the infected group, lymphocyte infiltration was seenespecially around the vessels and bile ducts in portal regions(data not shown).

Figure 1 shows the average FT-IR spectra of control livertissue in the 4000–450 cm�1 region. Spectra were normalizedwith respect to the amide A band located at 3300 cm�1. Theband assignments are presented in Table I. As seen from thefigure, the spectrum is quite complex, consisting of severalbands. The spectra belonging to the control and infected tissueare seen to be not identical. The spectral changes associatedwith these vibrational modes are of characteristic value, sincethey can provide valuable structural and conformationalinformation about the macromolecular constituents. As previ-ously mentioned in the determination of the mean values of thepeak positions, the band area, and the area ratio values, thespectrum belonging to each individual sample of the groupswere considered and these values are listed in Tables II and III.The relative information about the concentrations of the relatedfunctional groups was calculated from the area under the peaks.

The amide A band centered at 3300 cm�1 is mainly due tothe N–H stretching mode of proteins, polysaccharides, andintermolecular H bonding since free water was removed from

FIG. 1. An average FT-IR spectrum of mouse liver tissue in the 4000–450cm�1 region.

TABLE I. Major absorptions in IR spectra of control mouse liver tissue.

Peak no. Frequency (cm�1) Definition of the spectral assignment

1 3300 O–H stretching of proteins and intermolecular H bonding2 3014 Olefinic¼CH stretching vibration: lipids mainly unsaturated, cholesterol esters3 2959 CH3 asymmetric stretch: mainly lipid with a little contribution from proteins, carbohydrates, nucleic acids4 2926 CH2 antisymmetric stretch: mainly lipid with a little contribution from proteins, carbohydrates, nucleic acids5 2873 CH3 symmetric stretch: mainly protein with a little contribution from lipids, carbohydrates, nucleic acids6 2854 CH2 symmetric stretch: mainly lipid with a little contribution from proteins, carbohydrates, nucleic acids7 1650 Amide I (protein C¼O stretch)8 1541 Amide II (protein N–H bend, C–N stretch)9 1452 CH2 bending: mainly lipids, with a little contribution from proteins

10 1391 COO� symmetric stretch: fatty acids11 1238 PO2

� asymmetric stretch: mainly nucleic acids with a little contribution from phospholipids12 1151 CO–O–C asymmetric stretching: glycogen and nucleic acids13 1079 PO2

� symmetric stretching: nucleic acids and phospholipids. C–O stretch: glycogen14 1042 C–O stretching: polysaccharides15 969 C–Nþ–C stretch: nucleic acids

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the sample.30–35 The intensity and area of this band do notchange significantly for the infected liver sample (data notshown). Since the changes in the peaks are more dominant inthe C–H region, the detailed analysis was performed in thisregion.

The olefinic (¼CH) band at 3014 cm�1 arises from the CHstretching mode of the ¼CH groups and gives informationabout unsaturated lipids.19,23,34,35 As seen from Fig. 2, the peakarea of the 3014 cm�1 band in the infected group was seen tobe significantly higher than that of the control group, indicatingan increase in the content of unsaturated lipids. The peak areaof this band increased from 0.01 6 0.00 to 0.02 6 0.00 (p ,0.05) for the infected group. We have recently shown that thisband can be used for the rapid monitoring of lipidperoxidation.22 Therefore, the increase in olefinic band areais attributed mainly to the lipid peroxidation end products inthe infected tissues, as suggested previously for diabeticsamples.22 Infection causes inflammation in which inflamma-tory mediators take place. The breakdown of membranecomponents to form arachidonic acid can be responsible forthis lipid peroxidation.

The prominent peak at 2959 cm�1 is assigned to asymmetricCH3 stretching vibrations mainly due to lipids. In the infectedgroup the peak area of this band significantly decreased from0.71 6 0.09 to 0.50 6 0.16 (p , 0.05). This result shows thatthere is a change in the composition of the acyl chains.36 Thearea of the CH2 antisymmetric band at 2926 cm�1 alsodecreased from 2.49 6 0.25 to 2.16 6 0.42 (not statisticallysignificant) for infected samples. The decrease in the area of theCH2 antisymmetric stretching band at 2926 cm�1, which is oneof the major lipid bands, also indicates a decrease in theproportion of the CH2 groups. The other band at 2854 cm�1 isattributable to CH2 symmetric stretching, which is mainly dueto lipids. The area of this band also insignificantly decreased.The absorbance of the CH2 stretching vibrations are expectedto decrease when there is lipid peroxidation.37 These resultstogether with the significant variations of the olefinic bandimply that the infectious disease leads to lipid peroxidation inthe liver.

The ratio of the saturated lipids to unsaturated lipids wasobtained by taking the ratio of the sum of the areas of the CH3

asymmetric stretching and the CH2 symmetric stretchingmodes (2959 and 2926 cm�1, respectively) to the olefinicband at 3014 cm�1. This ratio decreased significantly from273.48 6 0.89 to 152.07 6 0.83 in the infected samples (p ,

0.01). The band at 2873 cm�1, which is assigned as CH3

symmetric stretching due to proteins, decreased in the infectedsamples (Fig. 3). The area of this band decreased from 0.08 6

0.01 to 0.05 6 0.02 (p , 0.05) for infected samples, whichreveals a decrease in the concentration of proteins.

From the FT-IR spectrum, a precise lipid-to-protein ratio canbe derived by calculating the area ratio of the CH2 symmetricstretching vibration (2854 cm�1) to the CH3 symmetricstretching (2873 cm�1) vibrations. As seen from Fig. 2, inthe infected groups, the area ratio of these bands increased from6.40 6 0.34 to 7.48 6 0.88 (p , 0.05). Generally, an increasein this ratio suggests a decrease in the protein content or an

TABLE II. The area values of the C–H stretching bands of the controland infected mouse liver tissue. The degree of significance was denoted as:p , 0.05*, p , 0.01**, p , 0.001***.

Frequency cm�1 Control Infected

3014 0.01 6 0.00 0.02 6 0.00*2959 0.71 6 0.09 0.50 6 0.16*2926 2.49 6 0.25 2.16 6 0.422873 0.08 6 0.01 0.05 6 0.02*2854 0.48 6 0.07 0.45 6 0.06

TABLE III. The area ratio values of the control and infected mouse livertissue. The degree of significance was denoted as: p , 0.05*, p , 0.01**, p, 0.001***.

Ratio Control Infected

2854/2873 6.40 6 0.34 7.48 6 0.88*2959/3014 58.85 6 0.52 32.34 6 0.85*2926 þ 2959/3014 273.48 6 0.89 152.07 6 0.83**

FIG. 2. The average FT-IR spectra of olefinic band (3014 cm�1) of control(–––) and infected (- - -) mouse liver tissues. The spectra were normalized withrespect to the amide A band.

FIG. 3. The average FT-IR spectra of control (–––) and infected (- - -) mouseliver tissues in the C–H stretching region (3050–2800 cm�1). The spectra werenormalized with respect to the amide A band.

APPLIED SPECTROSCOPY 201

increase in the lipid content or both.23,38 Since our findingsshowed that lipid content slightly decreased for infectedsamples, the increase in this ratio may be due to significantdecrease in the protein content. This was further supported by

the decrease in amide I and amide II bands, as can be seen fromFig. 4. The decrease in protein content might be due to proteindegradation in candida species resulting from a secretedaspartyl proteinase, which may be involved in tissue invasionby degrading extracellular matrix proteins, and secretedadenosine, which blocks neutrophil oxygen radical productionand degranulation.39 A critical function of inflammation is thedelivery of leukocytes to the site of injury. Leukocytes ingestoffending agents, kill bacteria and other microbes, and degradenecrotic tissue and foreign antigens. Unfortunately, leukocytesmay also prolong inflammation and induce damage byreleasing enzymes, chemical mediators, and toxic oxygenradicals.40 The variations in lipid content in infection can beexplained as the following: liver seems to use lipidsexcessively in order to fulfill its energy requirements, whichmay originate from the excess energy necessity of Kupffer cellsin immune response. The majority of Kupffer cells are found inthe liver periportal regions.41 Kupffer cells are unique cells fortheir role in both innate and acquired immunity. The primaryfunctions of Kupffer cells are phagocytosis, processing ofingested material, antigen presentation, and secretion ofbiologically active products.42

As can be seen from Fig. 3, no change was observed in thepeak positions of the CH2 symmetric and asymmetricstretching modes, which implies that the lipid order/disorder

FIG. 4. The average FT-IR spectra of control (–––) and infected (- - -) mouseliver tissues in the protein region (1900–450 cm�1). The spectra werenormalized with respect to the amide A band.

FIG. 5. Dendrogram presentation of control and disseminated candidiasis mice liver tissues. (A) 4000–450 cm�1, and (B) 1950–450 cm�1 region. The symbols areC: control and D: disseminated candidiasis.

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status was not changed in the case of the infection.43,44 Inaddition no change due to infection was observed in the lipidfluidity since no variation was obtained in the bandwidth ofCH2 stretching bands. No significant change was also observedin the secondary structure content of the proteins due toinfection using curve fitting analysis of the amide I band (Fig.4).

The FT-IR spectra were also subjected to cluster analysis.The analysis was performed in two different regions, namely4000–450 cm�1 (Fig. 5a) and 1900–450 cm�1 (Fig. 5b). As canbe seen from the figures, the dendrogram shows that most ofthe diseased samples (5 of 6) and all control samples (n ¼ 6)were grouped with the major cluster analysis. The dendrogramsshow that the normal (control samples) and diseased sampleswere divided successfully.

Invasive candidiasis, defined as candidemia and disseminat-ed candidiasis, is increasing in incidence. Infections caused byCandida spp. are the most common fungal infection inhospitalized patients. These organisms have been determinedto be the fourth most common cause of nosocomialbloodstream infection in the United States.3 The most commonCandida spp. isolated from cultures of liver biopsy samples isC. Albicans, as it is present in more than half of the patients.14

Various organs and systems can be affected with disseminatedcandidiasis.45 One of the most consistent biochemical findingsis a marked elevation of alkaline phosphatase, while other liverfunction tests such as bilirubin, aspartase aminotransferase(serum glutamic oxalacetic transaminase), and gamma glu-tamyl transferase can also show mild to moderate levels ofelevation. In histopathologic examination, macroscopically theliver is covered with yellowish nodules of 1–2 mm in diameter,and a well-defined granulamatous lesion with characteristicbudding yeasts with or without pseudohyphae is anotherexpected finding. However, liver biopsies do not alwaysprovide the best results because it is only in the very center ofthe lesion that fungal elements are found. Therefore, it isimportant to confirm the disease with other methods.

In conclusion, the current study revealed that the simulta-neous probing of the bands in the C–H region in FT-IR spectraprovides an easy, rapid, and sensitive monitoring of infectiousdisease-induced changes, as shown here with disseminatedcandidiasis. For this disease, changes in structural composi-tions such as an increase in unsaturation, a decrease in thesaturated lipid content, and an increase in the lipid/protein ratiocan be easily monitored by FT-IR spectroscopy. This studyshows that FT-IR spectroscopy is a valuable technique for theinvestigation of the pathology of infectious diseases andtherefore, it may be used for diagnostic purposes in infectiousdiseases.

ACKNOWLEDGMENTS

This work was partly supported by the research fund of Kocaeli Universityon the basis of a research project (No: 104/2001) and METU research fund.

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