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(Radiology. 2001;220:122-128.)
© RSNA, 2001

Experimental Studies

Cryptococcomas Distinguished from Gliomas with MR Spectroscopy: An Experimental Rat and Cell Culture Study1

Uwe Himmelreich, PhD, Theresa E. Dzendrowskyj, BA, Chris Allen, BSc, Susan Dowd, Richard Malik, BVSc, PhD, B. Philip Shehan, PhD, Peter Russell, FRACP, Carolyn E. Mountford, DPhil and Tania C. Sorrell, MD, BS, FRACP

1 From the Institute for Magnetic Resonance Research, Departments of Magnetic Resonance in Medicine (U.H., T.E.D., C.A., S.D., B.P.S., C.E.M.), Veterinary Clinical Sciences (R.M.), and Pathology (P.R.), and Centre for Infectious Diseases and Microbiology (T.C.S.), University of Sydney at Westmead Hospital, Rm 3114, Level 3, ICPMR, Darcy Rd, Westmead, New South Wales 2145, Australia. Received September 5, 2000; revision requested October 18; revision received December 8; accepted December 21. Supported by the National Health and Medical Research Council of Australia (grant 980116). Address correspondence to T.C.S. (e-mail: tanias@icpmr.wsahs.nsw.gov.au).


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
RESULTS
DISCUSSION
 REFERENCES
 
PURPOSE: To use magnetic resonance (MR) spectroscopy to characterize clinical isolates of Cryptococcus neoformans and a glioma cell line in culture and in experimental rats.

MATERIALS AND METHODS: One- and two-dimensional hydrogen 1 MR spectra were acquired from fungi cultured in vitro (16 isolates of C neoformans, three of Candida albicans, three of Aspergillus fumigatus, three of Saccharomyces cerevisiae) and a C6 glioma cell line. Cerebral biopsy specimens were obtained from healthy rats and animals with experimental infections or gliomas (19 healthy brains, 20 cryptococcomas, and 19 gliomas). Unequivocal signal assignment was performed for cell suspensions and tissue samples by using homo- and heteronuclear two-dimensional correlation spectra.

RESULTS: MR spectra of C neoformans and cerebral cryptococcomas—but not of other fungi, healthy brains, or gliomas—were dominated by resonances from the cytosolic disaccharide {alpha},{alpha}-trehalose. This spectral pattern was different from that of gliomas, which was dominated by lipids and an increased choline-creatine ratio, and that of healthy brain.

CONCLUSION: A remarkably high concentration of {alpha},{alpha}-trehalose in relation to other metabolites that are visible with MR spectroscopy is diagnostic of C neoformans. Cerebral cryptococcomas are an uncommon but serious manifestation of cryptococcosis in humans. Application of these results to the noninvasive diagnosis of cerebral cryptococcomas would help reduce the risk and expense of unnecessary surgery or biopsy and expedite patient treatment.

Index terms: Animals • Cryptococcosis, 10.2054 • Fungi, 10.2054 • Magnetic resonance (MR), experimental studies • Magnetic resonance (MR), spectroscopy, 10.12145 • Specimens, MR, 10.1261, 10.12145 • Specimens, MR spectroscopy, 10.12145 • Technology assessment


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
 REFERENCES
 
Cryptococcosis, caused by Cryptococcus neoformans, is a potentially life-threatening mycosis of immunocompromised and healthy hosts. C neoformans is the commonest cause of fungal meningitis (1), and circumscribed lesions (cryptococcomas) can occur in both lung and brain (2,3). Cerebral cryptococcomas have been reported in up to 14% of Australian patients with cryptococcosis, depending on host immune status at diagnosis (4). Brain lesions are usually diagnosed after C neoformans has been identified in tissue or fluids obtained from other body sites or in cerebrospinal fluid.

Brain biopsy is required for diagnosis when lesions are confined to the brain (3) or in the absence of other diagnostic material, since the pathologic characteristics of infective lesions cannot be reliably distinguished by using modalities such as computed tomography (5) or magnetic resonance (MR) imaging (6). Proton (hydrogen 1) MR spectroscopy offers the possibility of noninvasive diagnosis and has been applied to tumors, stroke, and bacterial infections (7–14). In vivo MR spectroscopy of the brain was developed and comprehensively tested for the diagnosis of human tumors (7,9,13) on the basis of initial ex vivo and in vivo studies in animal models (15). MR spectroscopy has been used to identify tumor pathologic findings in biopsy specimens in humans with a high sensitivity and specificity (16–20).

Compounds from micro-organisms and/or cells recruited during the host immune response that are visible with MR spectroscopy may give rise to diagnostic and prognostic markers. Extracellular carbohydrates and other products of C neoformans have been identified in cerebrospinal fluid in patients with cryptococcal meningitis (21). Cells of C neoformans are distinguished from those of other invasive fungal pathogens by the presence of an external polysaccharide capsule, which comprises a high percentage of the biomass in cryptococcomas. The purified capsular material has been studied by using 1H and carbon 13 MR spectroscopy (22). Investigators in more recent studies have identified extracellular products of C neoformans cultured in vitro by using MR spectroscopy (23).

To obtain adequate samples to generate statistically valid, robust algorithms for diagnosis, MR spectroscopy should initially be applied in micro-organisms cultured in vitro and in experimental animal models. The purpose of this study was to use MR spectroscopy to characterize isolates of the pathogenic fungus C neoformans and a glioma cell line in cultures and in experimental rats.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
DISCUSSION
 REFERENCES
 
In Vitro Cultures of Fungi and C6 Glioma Cell Line
Sixteen cryptococcal isolates were cultured in vitro and studied by using MR spectroscopy. These included eight clinical isolates of C neoformans serotype A (clinical isolates from lung, blood, cerebrospinal fluid, and brain), seven isolates of serotype B (clinical [brain and cerebrospinal fluid], veterinary, and environmental isolates), and one clinical isolate of the teleomorph of C neoformans, Filobasidiella neoformans var bacilliformis (32609; American Type Culture Collection, Manassas, Va). Other fungi that were cultured in vitro and studied by using MR spectroscopy included three each of the yeasts Candida albicans (clinical isolates) and Saccharomyces cerevisiae (environmental isolates and type cultures) and three of the fungus Aspergillus fumigatus (clinical isolates). Yeasts were identified biochemically (API 20C AUX system; BioMerieux, Marcy l’Etoile, France). Cryptococci were biotyped (24) and serotyped (Crypto Check agglutination test; Iatron Labs, Chiba, Japan).

Fungi were cultured for 24–48 hours on Sabouraud dextrose agar (Difco Labs, Detroit, Mich). Then they were cultured in either brain-heart infusion broth (Difco Labs) at 30°C (A fumigatus) or in yeast nitrogen broth (Difco Labs) containing 1% glucose, buffered at pH 7.0 with 0.345% wt/vol 3-(N-morpholino)propanesulphonic acid (Sigma Chemical, St Louis, Mo), at 27°C, 30°C, and/or 37°C. C6, a rat glioma cell line, was maintained as described previously (25) and used within three to 30 passages. Immediately before use, logarithmic phase fungal cells, or C6 glioma cells, were washed and resuspended in Dulbecco phosphate-buffered saline (Difco Labs) in animal models or in phosphate-buffered saline mixed with 99.5% D2O (Australian Nuclear Science and Technology Organization, Lukas Heights, Australia) for use at MR spectroscopy. Supernatants from growth media were suspended in D2O for use at MR spectroscopy.

Culture conditions for the growth of one isolate of C neoformans (McBride strain) were varied to test the effect of stress on MR-visible metabolite profiles. The isolates were cultured in buffered yeast nitrogen broth containing 10 mmol/L of glucose. The following parameters were varied: incubation temperature (27°C, 35°C, 42°C), pH level (pH 5.0 or 7.0), glucose concentration (1, 10, 50 mmol/L), substitution of glucose with mannose (10 mmol/L) or sucrose (10 mmol/L), and prolonged incubation in glucose-free medium (0–100 hours).

Animal Studies
Three isolates of C neoformans serotype B (WM276, WM430, McBride) and the C6 glioma cell line were used for animal experiments. Male Wistar-Furth and female Fischer 344 rats (Animal Research Council, Perth, Australia; weight, 150–250 g) were anesthetized by means of inhalation of 4% halothane in 100% oxygen prior to intraperitoneal injection of ketamine hydrochloride (Apex Laboratories, Sydney, Australia; 11.6 mg per kilogram of body weight) and xylazine hydrochloride (Apex Laboratories; 1.2 mg/kg) and were allowed to spontaneously breathe room air. For induction of brain lesions, the animal’s head was fixed in a stereotactic frame (David Kopf Instruments, Tajunga, Calif); 5 µL of a suspension of C neoformans serotype B, or C6 glioma cells, was injected through a straight flat-ended 26-gauge needle at a rate of 3–6 µL/min. Preliminary experiments established that 5 x 104 colony-forming units of cryptococci suspended in a volume of 5 µL, and 1 x 106 C6 cells, also in a volume of 5 µL, induced lesions of at least 3 mm in diameter when harvested 6–12 days (cryptococcomas; rats, n = 18) or 12–30 days (gliomas; rats, n = 26) postoperatively.

Optimal coordinates for microinjection were 2.0 mm below the dura and 3.0 mm lateral and 2.4 mm anteroposterior relative to the ear bar of the stereotactic frame, which functions as the zero line. By using these coordinates, rats were injected for use in the MR study with the C neoformans serotype B isolate McBride (cryptococcomas, n = 20; gliomas, n = 19; controls, n = 19). Control tissue was obtained from saline-injected rats. At appropriate times, the rats were sacrificed, and the brain was removed and cut transversely at the site of the lesion. Brain tissue was fixed in formalin and embedded in paraffin; 7-µm sections were taken and stained with hematoxylin-eosin or periodic acid-Schiff reagent for light microscopy. Brain tissue samples (maximum diameter of 4 mm) from each of the animals with cryptococcomas or gliomas and from controls were suspended in phosphate-buffered saline with D2O, snap-frozen in liquid nitrogen, and stored at -70°C for as long as 4 months for use at MR spectroscopic analysis.

Animal experimentation was carried out according to the Australian National Health and Medical Research Council Guidelines and with ethical approval from the University of Sydney Animal Ethics Committee.

MR Experiments
1H MR spectra were obtained with a 360-MHz spectrometer (Bruker Avance; Bruker, Rheinstetten, Germany) equipped with a 5-mm {1H, 13C} inverse-detection dual-frequency probe. The temperature was maintained at 37°C. Residual water signal was suppressed with selective gated irradiation (26) or selective excitation by using pulse field gradients (27). Chemical shifts were referenced to external sodium 3-(trimethylsilyl) propanesulfonate at 0 ppm or internal water at 4.65 ppm. One-dimensional (1D) 1H MR spectra were acquired with a spectral width of 3,600 Hz, a time domain of 8,192, 128 or 256 acquisitions, and a relaxation delay of 1 second. A line broadening of 1 or 3 Hz was applied for cell culture or tissue samples, respectively, prior to Fourier transformation. Resonance ratios from fully relaxed 1H MR spectra were used for comparison of cell types. Packed-cell suspensions and samples were spun at 20 Hz to prevent the cells from settling in the MR tube. A relaxation delay of 5 seconds was applied to allow full relaxation.

Two-dimensional (2D) MR spectra were acquired for unequivocal signal assignment. {1H, 1H} COSY experiments were performed in magnitude mode (28). Acquisition parameters were a sweep width in time domain in the second dimension (t2) of 3,600 Hz; a t2 of 2,048, with 256 increments of 32 or 48 acquisitions each; and a relaxation delay of 1 second. Sine-bell window functions were applied in the time domain in the first dimension (t1), and Gaussian-Lorentzian window functions were applied in t2. Zero filling was used to expand the data matrix to 1,024 in t1. Cross-peak volumes were determined as previously described (29).

TOCSY spectra with mixing times of 40 and 120 msec were acquired with 256 increments of 2,048 data points and 48 acquisitions per increment for confirmation of assignments (30).

{1H, 13C} One-bond shift-correlation spectra were obtained in the 1H detection mode by using an HSQC pulse sequence (31) for some samples to confirm signal assignments. The 1H MR spectral width was 3,600 Hz, and the 13C MR spectral width was 15,000 Hz. 13C MR decoupling during acquisition was achieved by using globally optimized alternating-phase rectangular pulses 1 (32). The evolution time (t1) was incremented to obtain 256 free induction decays, each consisting of 80 acquisitions and 2,048 data points. The relaxation delay was 1 second. A sine-bell function was applied in t2, and a Gaussian-Lorentzian function was applied in t1. Zero filling to 1,024 points was used in t1 prior Fourier transformation.

1H MR spectra and 2D {1H, 1H} COSY MR spectra were acquired for all fungal isolates, the C6 glioma cell line, and rat brain samples (20 with cryptococcomas, 19 with gliomas, and 19 controls). Signal assignment was confirmed by using TOCSY and HSQC for at least one isolate or sample in each of the four fungal species, the C6 glioma cell line, and the brain biopsy samples.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
DISCUSSION
 REFERENCES
 
Cell Culture Studies
Typical 1D and 2D MR spectra of fungi cultured in vitro and the C6 glioma cell line are compared in Figure 1. Major cross-peaks from the 2D COSY spectra are summarized in Table 1. Resonances were assigned either by means of comparison of findings with resonances in the literature (8,16,23,25,33) or primary analysis of COSY, TOCSY, and HSQC spectra. Resonances listed in Table 1 and shown in Figure 1 were present in spectra of all isolates of the respective fungi and the C6 glioma cell line. Resonance intensities varied among isolates as shown in Table 2.



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  		Figure 1. One-dimensional 1H MR spectra of in vitro cell cultures. A, C neoformans. Note the prominent trehalose resonances, which are not distinguishable on the other spectra. B, C albicans. C, A fumigatus. D, S cerevisiae. E, C6 cell line. AA, amino acids; ac, acetate; CH, nonspecific carbohydrate resonances; H1-H6, 1H MR resonances from {alpha},{alpha}-trehalose (tre); lip, lipids; N(CH3)3, contributions from choline-containing compounds (choline, phosphocholine, glycerophosphocholine), betaine, and taurine; NCHn, contributions from creatine, {gamma}-aminobutyric acid, and lysine residues.

 

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  		TABLE 1. Major Components in the COSY Spectra Identified by Their Cross-Peak Volumes

 

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  		TABLE 2. Peak Ratios for Selected 1H MR Signals

 
C neoformans Cultured in Vitro
One- and two-dimensional COSY MR spectra of C neoformans were dominated by resonances from lipid and {alpha},{alpha}-trehalose. The spectral pattern of lipids showed resonances at 0.90, 1.30, 1.60, 2.00, 2.30, and 5.38 ppm (25). Resonances at 3.46, 3.66, 3.77–3.85, and 5.19 ppm were assigned to {alpha},{alpha}-trehalose by using HSQC spectroscopy on the cell suspensions. The respective assignments were as follows: H1-C1 (5.19–93.5 ppm), H2-C2 (3.66–71.5 ppm), H3-C3 (3.86–73.0 ppm), H4-C4 (3.46–70.0 ppm), H5-C5 (3.83–72.5 ppm), and H6-C6 (3.78 and 3.88–61.0 ppm). Less intense cross-peaks from amino acid residues (lysine, alanine, threonine, and glutamate and/or glutamine) and ethanol were evident in some strains (Tables 1 and 2). The dominant MR-visible extracellular metabolites were acetate (1.92 ppm) and ethanol (1.16 and 3.63 ppm).

Effect of Stress on Cryptococcal Cells
Since trehalose has been reported to protect fungi against adverse conditions (eg, heat, desiccation, osmotic and oxidative stress) (34–38), we considered the possibility that the MR-visible trehalose could vary with culture conditions. We tested the effect of temperature, pH, glucose concentration, substitution of glucose with other sugars, and incubation time as specified in Materials and Methods. The respective resonance ratios varied by no more than a factor of four relative to standard conditions. Lipid and trehalose signals remained dominant in 1D and COSY spectra, irrespective of culture conditions.

MR Spectroscopy of Other Fungi Cultured in Vitro
MR spectroscopy findings in two other clinically important pathogenic yeasts, C albicans and S cerevisiae, and the fungus A fumigatus were investigated and found to be different from those of C neoformans. The 1D and 2D COSY spectra of C albicans (Fig 1, B) and S cerevisiae (Fig 1, D) revealed dominant lipids, whereas those of A fumigatus (Fig 1, C) were characterized by resonances from amino acid residues and carbohydrates. Carbohydrate resonances from these fungi were of a much lower intensity than those from C neoformans and could not be assigned to specific monosaccharide residues. Small amounts of MR-visible {alpha},{alpha}-trehalose (approximately 20 times less than in C neoformans) were identifiable only in the COSY spectra of two of the three strains of S cerevisiae and in none of C albicans, if exposed to high temperatures (37°C–43°C). Ethanol was identified in the two yeast species. Acetate was visible in the spectra from the culture supernatants from C albicans and A fumigatus.

C6 Glioma Cell Line
The spectra of suspended C6 glioma cells cultured in vitro (Fig 1, E; Tables 1, 2) were dominated by resonances from amino acids. The 2D spectra of C6 cells revealed no carbohydrate cross-peaks. Intense cross-peaks arising from choline, phosphocholine, and glycerophosphocholine and relatively high amounts of taurine and the amino acid residues leucine and glutamate and/or glutamine were present, as has been reported (25) for tumor cell lines. The resonance ratio of 3.25:3.05 ppm, representing choline- and creatine-containing compounds, was higher than that in the spectra of fungi (Table 2).

Animal Studies
Histopathologic findings in biopsy samples of rat brains showed that the biomass of cryptococcomas was composed predominantly of cryptococci, as is seen in human infection, and verified the pathologic findings in tumors grown in the rat model. Representative 1D 1H MR and 2D COSY spectra of control rats, rats with cerebral cryptococcoma, and rats with gliomas are shown in Figure 2. Resonance ratios are summarized in Table 2.



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  		Figure 2. One- and two-dimensional COSY MR spectra of cerebral rat tissue samples. A, Brain tissue of control rat. B, Tissue infected with C neoformans. C, Tissue with glioma. Points A-D and F, triglyceride resonances (25); AA, amino acid residues; ac, acetate; ala, alanine; chol, choline; eth, ethanol; gaba, {gamma}-aminobutyric acid; glu/gln, glutamate and/or glutamine; h-tau, hypo-taurine; lac, lactate; leu, leucine; lip, lipid; lys, lysine; mI, myo-inositol; NAA, N-acetyl aspartate; NCHn, contributions from creatine, phosphocreatine, {gamma}-aminobutyric acid, and lysine; N(CH3)3, contributions from choline-containing compounds (choline, phosphocholine, glycerophosphocholine), betaine, and taurine; PC, phosphocholine; PE, phosphoethanolamine; tau, taurine; tre, trehalose. Note that the listed amino acids refer to amino acid residues and not necessarily to the respective free amino acids.

 
Spectra of brain tissue in controls were dominated by N-acetyl aspartate at 2.0 ppm. Characteristic signals of lower intensity included composite peaks at 2.0–2.2 ppm (glutamine and/or glutamate), at 3.0 ppm (creatine, phosphocreatine, {gamma}-aminobutyric acid, and lysine residues), at 3.2 ppm (eg, N[CH3]3 groups of choline, phosphocholine, and glycerophosphocholine), and at 3.6–3.9 ppm (H{alpha} of amino acid residues and myo-Inositol). myo-Inositol and amino acid ({gamma}-aminobutyric acid, choline, phosphocholine, and glycerophosphocholine) signals were also present in COSY spectra. Lactate signals of variable intensity, resulting from anaerobic metabolism occurring in the time between excision and freezing, were found at 1.3 ppm.

Cryptococcoma yielded 1D and COSY MR spectra with the typical pattern of cryptococcal trehalose and lipids described previously. The intensity of the lipid resonance at 5.38 ppm to that of the trehalose resonance at 5.19 ppm varied over a wide range (Table 2). Furthermore, the 3.25:3.05-ppm resonance ratio was increased, compared with that of normal brain tissue. The N-acetyl aspartate signal decreased dramatically and was undetectable in some samples. Other resonances not observed in normal brain tissue arose from acetate (1D spectra) and ethanol (2D COSY) in some but not all spectra. Also, a distinct cross-peak arose from glycerophosphocholine, which was of much higher intensity than in control and glioma spectra. The myo-inositol and {gamma}-aminobutyric acid cross-peak intensities in the COSY spectra were reduced relative to the amino acid cross-peaks when compared with those of brain tissue in controls.

Spectra of tumor biopsy samples were dominated by lipid signals and an increased resonance ratio at 3.25:3.05 ppm, which is consistent with many reports (8,39,40) in the literature. The relative increase in lipid signal intensities and the increase in the 3.25:3.05-ppm ratio was, for most samples, much larger than the increase found in cryptococcomas (Table 2). Resonance ratios for the lipid varied. N-acetyl aspartate remained undetectable in many tumor specimens, indicative of absent neuronal activity. The only cross-peaks apart from lipids that increased relative to other amino acid residue cross-peaks (eg, lysine, leucine) were those of taurine (3.28–3.50 ppm), choline (3.50–4.07 ppm), phosphocholine (3.61–4.19 ppm), and phosphoethanolamine (3.22–3.98 ppm).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
 
C neoformans was distinguished unequivocally from other yeasts, the filamentous fungus A fumigatus, and C6 glioma cells by using MR spectroscopy, due to an abundance of the nonreducing disaccharide trehalose. These differences were recorded in spectra of cells cultured in vitro, as well as in those of affected tissue from rat cortex, in which the diagnosis was confirmed histologically. MR spectroscopy has thus provided a means of distinguishing cryptococcomas from healthy brain and brain tumor tissue in biopsy samples.

C neoformans in infected tissue is surrounded by a capsule that typically occupies many times the volume of the fungal cell. This capsule is composed predominantly of glucuroxylomannans in a loosely woven fibrillar configuration (21,22). None of the cell-associated glucuroxylomannans from samples cultured in vitro, or from tissue biopsy samples, were MR-visible, indicating that native capsular polysaccharides are not sufficiently mobile to be visible with the use of MR spectroscopy.

In contrast, the cytosolic compound trehalose was identified and was present in large amounts. Trehalose is present in yeasts and other fungi (41) and is, therefore, not a unique characteristic of C neoformans, per se. It is an important protectant induced by conditions of heat (38), osmotic stress (42), dehydration (37), desiccation, and other factors (for review, see references 35 and 36). Trehalose levels in C neoformans cultured at 25°C, however, exceeded those in S cerevisiae under conditions of heat stress (37°C) by at least 20 times. Altered culture conditions did not reduce the intensity of the trehalose signals in C neoformans.

The large amount of trehalose relative to other MR-visible compounds on the spectra of C neoformans defines trehalose as one marker that can be used to distinguish C neoformans from other fungi. It is possible that such high levels of trehalose in cryptococci are an evolutionary response to environmental stress, particularly temperature, dehydration, and starvation. Adaptation to survival and growth at physiologic temperatures, a recognized virulence determinant of C neoformans (43) is consistent with high intracellular concentrations of trehalose.

Bacterial metabolites, but not {alpha},{alpha}-trehalose, have been identified by using 1H MR spectroscopy in pus samples from patients with bacterial brain abscesses (10,11, 15,44). Ethanol, a product of glucose fermentation in yeasts, was reported (45) to be present in the cerebrospinal fluid of a patient with cryptococcal meningitis. The predominant extracellular metabolites (ie, acetate, ethanol) found in the present study and in that by Bubb et al (23) are not suitable for definitive in vivo or ex vivo diagnosis of cryptococcomas, since they are also produced by other pathogenic micro-organisms (44). The distinctive acetate signal present in many cryptococcomas, but not in healthy or neoplastic tissue, may, however, be a useful diagnostic indicator of infection. Acetate is produced by bacteria and has been identified in bacterial abscesses (10,11,15), as well as in C neoformans and cryptococcomas in this study, by using MR spectroscopy.

Practical application: MR spectroscopy can be used to distinguish unequivocally between healthy brain and experimental cryptococcomas and gliomas in rats. The high level of MR-visible {alpha},{alpha}-trehalose recorded from cryptococcomas provides a basis for the pathologic diagnosis of cerebral cryptococcomas. It now remains to be determined if this method is applicable to in vivo diagnosis of cerebral cryptococcomas in humans. If so, cerebral cryptococcomas might no longer be mistaken for malignancies with the use of conventional imaging modalities. An early and correct diagnosis will reduce the high morbidity and mortality rates (3) that occur when diagnosis is delayed. Introduction of in vivo MR spectroscopy as a noninvasive method of diagnosis of infective lesions in the brain will reduce the risk and expense of unnecessary surgery or biopsy and expedite patient treatment decisions.


   ACKNOWLEDGMENTS
 
We thank Thomas H. Ng, PhD, for reporting on the histopathologic findings and Scott McDonald, Russell Banks, BSc, and Heide-Marie Daniel, MSc, for technical assistance.


   FOOTNOTES
 
Abbreviations: 1D = one-dimensional, 2D = two-dimensional

Author contributions: Guarantors of integrity of entire study, T.C.S., U.H.; study concepts, C.E.M., T.C.S.; study design, C.E.M., B.P.S., T.C.S.; literature research, T.E.D., C.A.; clinical studies, R.M., P.R.; experimental studies, S.D., T.E.D., C.A., B.P.S., U.H.; data acquisition and analysis/interpretation, T.E.D., C.A., U.H.; statistical analysis, U.H.; manuscript preparation, U.H., T.E.D., T.C.S.; manuscript definition of intellectual content and editing, U.H., T.C.S., C.E.M.; manuscript revision/review, T.C.S., C.E.M.; manuscript final version approval, U.H., T.C.S.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
DISCUSSION
 REFERENCES
 

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