INFECTION
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IMMUNITY, Mar. 1978, p. 992-998
Vol. 19, No. 3
0019-9567/78/0019-0992$02.00/0 Copyright © 1978 American Society for Microbiology
Printed in U.S.A.
Biochemical Examination of Sera During Systemic Candida Infection in Mice DONNA OBLACK,1 JAN SCHWARZ,2 AND IAN A. HOLDER3* Departments of Microbiology' and Pathology, University of Cincinnati College of Medicine, and Shriners Burns Institute,3 Cincinnati, Ohio 45219 Received for publication 6 July 1977
Candida pathogenesis was examined by intravenous challenge of mice with either C. albicans or C. guilliermondii. Animals were moribund 12 h postchallenge with C. albicans and were found to have the greatest number of organisms in the heart and kidney, severe interstitial myocarditis, and elevated serum levels of blood urea nitrogen, creatine phosphokinase, serum glutamic oxalacetic transaminase, serum glutamic pyruvic transaminase, and lactic dehydrogenase. In contrast, challenge with C. guilliermondii resulted in a significantly lower renal census, no myocarditis, and no significant change in the concentration of these serum constituents. Challenge with nonviable C. albicans did not produce the effects observed with viable organisms. Moreover, challenge with filamentous C. albicans resulted in biochemical alterations of lower magnitude and in lower mortality rates. These results indicated that altered serum biochemistries were correlated with the histopathology of fatal Candida infection and that there were distinct differences with C. guilliermondii and the dimorphic phases of C. albicans.
genus Candida, one pathogenic and the other nonpathogenic, were used in these studies to enable recognition of those events that are unique to the pathogenic organism. Using the biochemical alterations described, we examined several variables for their effect on Candida pathogenicity. (These data were presented in part at the 76th Annual Meeting of the American Society for Microbiology, Atlantic City, N.J., 2-7 May
The frequency of deep fungal infection has increased steadily during the past 20 years, with yeasts of the genus Candida emerging as a very common cause of fungal infection of compromised hosts (2, 3, 11, 33, 37). Our knowledge of the events occurring during systemic Candida infection is mainly limited to descriptions of the host response against the yeast. For example, several investigators have examined the histopathology of systemic C. albicans infection. Postmortem examinations of human tissues have shown the kidney to be the organ most frequently containing lesions (15, 23, 30). In addition to renal involvement, at least 50% of patients were found to have lesions in the heart (15, 20, 23, 30, 37). Similarly, in murine models of C. albicans infection, the involvement of the kidney has been noted frequently (13, 16, 21, 28). Descriptions of gross and microscopic examination of this organ cite extensive abscess formation in which both yeast cells and pseudohyphae are present. In some cases (1, 6, 16, 19, 21, 42), the heart also has been involved with myocarditis and abscess formation. The heart has not been cited in all reports, however, so the frequency of its involvement may be underestimated. This study was undertaken to identify biochemical alterations which reflect the histopathology of experimental systemic C. albicans infection in mice. Two different species of the
1976.) MATERIALS AND METHODS Mice. Female, outbred Swiss mice, strain CD-1 (Charles River, Wilmington, Mass.), weighing 22 to 24 g were used in all experiments. Preparation of inocula. The two species of Candida utilized, C. albicans strain CA 2 and C. guilliermondii strain F 17, were obtained from the Mycology Laboratory of the Cincinnati General Hospital. Organisms were grown for 17 h at 370C in Sabouraud dextrose broth (SDB), harvested by centrifugation, and washed three times in sterile distilled water. Inocula for injection were adjusted to the standard concentration by use of a hemacytometer. Dilutions of inocula plated on Sabouraud dextrose agar (SDA) plates in triplicate showed that greater than 98% of the cells were viable. A 0.1-ml amount of the culture suspension was injected into a lateral tail vein. Animals were challenged in groups of 10. Nonviable cells were prepared by heating suspensions of C. albicans in a water bath at 560C for 90 to 120 min. Samples of the heat-treated suspensions were 992
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plated on SDA plates to confirm nonviability. Histology. At 12 h postinfection, organs were excised and fixed for 24 h in 10% buffered Formalin. Duplicate sections were prepared from each organ. One section was stained with hematoxylin-eosin (24), and the other was stained with methenamine-silver (10) with hematoxylin-eosin counterstain. Colony counts. The numbers of yeasts in various organs of infected animals were determined 12 h postchallenge by the method of Saymen et al. (39). In each experiment, the like organs from three animals were weighed and then homogenized as one sample. The number of yeasts in each homogenate was determined by dilution in SDB and plating on duplicate SDA plates. Plates were incubated at 37°C for 48 to 72 h. The total count in each organ was expressed as the number of colony-forming units (CFU) per gram (wet weight) of organ. Numbers of CFU are reported in log units as the mean of three experiments. Serum chemistries. Groups of animals were anesthetized with methoxyfluorane, and 100 to 150 ,tl of blood was obtained from the retro-orbital sinus 11 h prior to challenge with either organism; controls were injected with the diluent alone. A second blood sample was collected from each animal 11 to 12 h postinjection. A Clinicard 368 Analyzer (Instrumentation Laboratory, Inc., Lexington, Mass.) was used to analyze 25 ,ul of individual sera for the concentration of blood urea nitrogen (BUN) by the direct assay method of Crocker (7). From a control group and the two Candida-challenged groups of animals, other blood samples were obtained by cardiac puncture 12 h postinjection, and 100-,ul amounts of individual sera were analyzed for the concentration of creatine phosphokinase on a Clinicard 368 Analyzer. This methodology is based on the coupled enzymatic method of Oliver (29) with the modifications made by Rosalki (36) and Hess (14). Levels of other serum constituents were determined on pooled sera obtained from groups of 20 to 30 animals 12 h postchallenge with Candida. Sera from 20 uninfected mice were pooled and used as a control. Serum electrolytes (sodium, potassium, chloride) and carbon dioxide in the sera were determined by use of Stat/Ion Instrumentation (Technicon Corp., Tarrytown, N.Y.). Other serum constituents (total protein, albumin, calcium, phosphorus, glucose, uric acid, creatinine, total bilirubin, alkaline phosphatase, lactic dehydrogenase, and serum glutamic oxalacetic transaminase) were analyzed by use of a Sequential Multiple Analyzer-12 (Technicol Corp.). The concentration of serum glutamic pyruvic transaminase was determined by the change in ultraviolet absorbance at 340 nm with a Centrifichem System 400 (Union Carbide Corp., Rye, N.Y.). Statistical analysis. Student's t test was used to analyze the variance between experimental groups. Data were considered significant at a level of P < 0.025. Mortality studies of the dimorphic phases of C. albicans. Blastospores of C. albicans were harvested after 18 to 22 h of growth on SDA plates at 37°C and were inoculated into heat-inactivated normal human sera. Upon incubation at 37°C, these cells undergo transition to a filamentous phase (32, 41). In contrast,
993
similar cultures maintained at 250C retain the yeastphase morphology. Cells were collected after 30, 60, or 180 min of incubation at the two temperatures, washed, and injected intravenously into groups of 9 or 10 animals. The results are expressed as the percent mortality during the experimental period. The difference between experimental groups was analyzed with the chi-square test (31).
RESULTS Pathogenicity of Candida. Intravenous challenge of mice with 106 C. albicans cells resulted in a rapidly lethal infection. This dosage was completely lethal (LDioo) in 3 days. Within the first 24 h, 50% of the experimental group died, and the moribund appearance of mice 12 h postchallenge was most striking. In contrast, no deaths were recorded in 28 days when mice were challenged with equivalent numbers of C. guilliermondii cells. Colony counts. The organ populations of Candida 12 h postchallenge are presented in Table 1. The greatest numbers of CFU of C. albicans were found in the heart and kidney of the moribund animals. When animals challenged with the two species were compared, the numbers of CFU were equivalent in the brain, heart, liver, and lungs. Statistically significant greater numbers of C. albicans than C. guilliermondii were found in the kidney; in one organ, the spleen, C. guilliermondii was present in number one log higher than C. albicans. Histology. Histopathological examination of heart tissue 12 h postchallenge revealed severe focal myocarditis only in those animals challenged with C. albicans. Numerous foci of myocarditis showing microabscesses and tissue destruction were observed. Using the methenamine-silver stain, we observed yeast cells and pseudohyphae of C. albicans in these lesions. There was complete absence of inflammation in the myocardium of animals challenged with C. TABLE 1. Organ populations 12 h after intravenous challenge with Candida Logio CFU/g (wet wt) of organ Organ C. albicans
C. guilliernondii
4.12 ± 0.16 Brain .4.53 ± 0.04a 6.02 ± 0.14 Heart .6.06 ± 0.19 5.93 + 0.09 5.22 ± 0.09b Kidney .. 5.36 ± 0.08 Liver .5.52 ± 0.08 5.59 ± 0.09 Lung .5.61 ± 0.04 4.86 ± 0.11 5.83 + 0.25b Spleen aMean ± standard error. Each value represents three experiments, each comprising three animals. b p < 0.025, calculated from Student's t test comparing the groups challenged with C. albicans and C.
guilliermondii.
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INFECT. IMMIJN.
OBLACK, SCHWARZ, AND HOLDER
Although large numbers of CFU of C. albihad also been recovered from kidney homogenates 12 h postinfection, a cellular infiltrate was not present in the kidney at this time. BUN. Abnormalities of kidney function were observed in mice 12 h postinfection with the yeast phase of C. albicans (Table 2). The BUN of animals challenged with C. albicans was significantly higher than that of uninfected controls. Challenge with an equivalent or 10-fold higher number of C. guilliermondii cells did not result in an elevation of the BUN. Challenge with 106 or 107 nonviable C. albicans cells was also without effect in this regard. BUN elevation was observed only when animals were challenged with yeast-phase cells of C. albicans; intravenous injection of equivalent numbers of filamentous cells did not produce a significant change. In two experiments, the serum BUN level after Candida challenge was significantly lower than that observed prior to challenge when both samples were collected from the same animal (Table 2, see BUN data with 106 C. guilliermondii and 106 nonviable C. albicans cells). Collecting two blood samples from uninfected control animals also resulted in a significantly lower BUN in the second sample. To avoid this artifact of significantly lowered BUN levels resulting from multiple bleedings, blood samples from uninfected and infected animals in the other experiments were collected from different animals. Creatine phosphokinase. Sera from animals challenged with both Candida species were analyzed for the concentration of creatine phosphokinase, an enzyme localized in heart, skeletal muscle, and brain (34) which is released in quantity into the circulation after cellular destruction
in those tissues. The data presented in Table 3 indicate that acute injury of myocardial tissue, consistent with the histopathological evidence, occurs during systemic infection with C. albicans. The serum creatine phosphokinase level of mice, 12 h postchallenge, was elevated 30-fold over controls. That of mice challenged with a comparable or 10-fold higher number of C. guilliermondii cells was not significantly different from control animals. Elevated serum creatine phosphokinase levels were not observed when animals were challenged with nonviable C. albicans, either at the same dosage or at a 10-fold greater dosage. Furthermore, nonviable cells did not elicit a cellular infiltrate in the myocardium of these animals. When animals were challenged with filamentous C. albicans, creatine phosphokinase was elevated to an intermediate level (about fourfold, Table 3). Challenge with 106 or 107 nonviable filamentous cells did not result in significantly elevated creatine phosphokinase levels. Electrolytes and other serum chemistries. To document other biochemical alterations during Candida infection, pooled sera from uninfected controls and from animals challenged with either C. albicans or C. guilliermondii were assayed for the concentration of 16 additional components. The serum concentrations of 13 of the 16 additional components were equivalent in the three groups of animals. However, the concentrations of three enzymes in the sera of animals challenged with C. albicans were found to be elevated twofold or greater over that of uninfected controls (Table 4). At 12 h after challenge, there was a fivefold increase in lactic dehydrogenase, an eightfold increase in serum glutamic oxalacetic transaminase, and a threefold increase in serum glutamic pyruvic transaminase. Elevated serum concentrations of these enzymes were not observed in animals after challenge with C. guilliermondii.
TABLE 2. Evaluation of kidney function 12 h after intravenous challenge with Candida
TABiF, 3. Evaluation of cardiac injury 12 h after intravenous challenge with Candida Creatine phosphokinase (IU/liter)
guilliermondii despite the presence of equivalent numbers of organisms documented in Table 1. cans
BUN (mg/100 ml)
Challenge
Challenge
Uninfected
Candida chal-
controls
lenged
C. albicans cells 106 cells, nonviable l07 cells, nonviable
106
106 cells, filamentous
29.2 + 1.4 (1O)" 32.5 + 1.0 (10) 24.2 1.2 (10) 28.5 1.5 (10)
51.9 26.2 24.8 27.8
22.7 28.2
± ± ± ±
5.4b (10) 1.2 1.0 0.8
(10) (10) (10)
C. guilliermondii
106 cells 107 cells
29.7
±
1.0 (15)
27.6
±
1.5 (10)
± ±
1.9 (9) 1.7 (9)
aMean ± standard error. The numbers in parentheses represent total animals tested; each animal was assayed once. b < 0.025, calculated from Student's t test comparing uninfected controls and Candida-challenged groups.
Uninfected controls
Candida challenged
C. albicans 10" cells 248 ± 40 (8)" 7,908 ± 1,002" (10) 10" cells, nonviable 182 ± 19 (10) 193 ± 21 (10) 107 cells, nonviable 285 ± 23 (8) 331 ± 34 (7) ± 10" cells, filamen- 285 27 (9) 1,069 ± 81" (10) tous C. guilliermondii 10' cells 395 + 80 (10) 292 ± 59 (10) 10' cells 205 ± 31 (9) 346 ± 66 (10) " Mean ± standard error. The numbers in parentheses represent total animals tested; each animal was assayed once. P < 0.025, calculated from Student's t test comparing uninfected controls and Candida-challenged groups.
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TABLE 4. Serum enzyme levels 12 h after intravenous challenge with 10U Candida cells Enzyme
Uninfected controls
C. albicans challenged
C. guihallerinond
challenged
560 522-600a 2,400 Lactic dehydrogenase .... 135 115-124 1,000 Glutamic oxalacetic transaminase 20 49 18 Glutamic pyruvic transaminase " Enzyme concentration in milliunits per milliliter. Each value given represents assay of pooled sera collected from 20 to 30 animals.
Virulence of the dimorphic phases of C. tive examinations of Candida growth in vivo albicans. The previous data, indicating that have been only infrequently reported. Kemp and yeast-phase cells produced greater elevations of Solotorovsky (19) investigated Candida infeccreatine phosphokinase and BUN than filamen- tion in mice and reported increasing CFU in the tous cells, prompted investigation of the differ- heart and kidney during the early stages of the infection. Louria et al. (21) found high numbers ences in virulence between the two phases. Filamentation of C. albicans in serum is a of CFU of C. albicans in the kidneys of mice species-, substrate-, time- and temperature-de- when moribund within 6 days after challenge. It pendent phenomenon (41). Figure 1 depicts the has recently been reported that, on a weight progression of filament formation by C. albicans basis, the kidney clears a greater percentage of upon incubation in serum for various times at the Candida inoculum than the liver, lungs, or 370C. At the time of inoculation, only yeast- spleen (35). Whether this capacity contributes phase cells were present (Fig. 1A). However, to the ultimate growth of C. albicans in the within 30 min of incubation, an occasional yeast kidney remains to be determined. One unexcell exhibited the beginning of the filament (Fig. pected finding of the quantitation experiments 1B). After 1 h of incubation more than two- was the observation of 10-fold greater CFU of C. thirds of the yeast cells had formed short fila- guilliermondii than of C. albicans in spleen ments (Fig. 1C), and after 3 h of incubation homogenates 12 h postchallenge. Inasmuch as greater than 90% of the yeast cells had filaments neither Candida species multiplies in the spleen (Fig. 1D), most of which were longer than those (9, 19, 27, 35), this observation may have little observed at 1 h. Yeast cells inoculated into se- bearing on Candida pathogenesis. Although myocarditis has not been regularly rum and maintained at 250C for comparable emphasized in Candida infection, our observatimes retained a yeast morphology. Presented in Table 5 are the mortalities ob- tion of myocarditis occurring acutely in the served after intravenous challenge with 106 cells course of fatal Candida infection supports the of the dimorphic phases of C. albicans incubated work of several investigators (1, 6, 16, 21). This for 30, 60, or 180 min in serum. As the transition response was specifically produced by C. albifrom the yeast to the filamentous phase pro- cans, for, despite the presence of equivalent numbers of CFU in the myocardium 12 h postceeded, a decrease in mortality occurred. The populations of C. albicans in various challenge, a cellular infiltrate was not elicited by organs 12 h after challenge with 106 filamentous C. guilliermondii. Histological examination of cells are presented in Table 6. The greatest kidney sections indicated that a cellular host number of CFU were found in the kidney. When response occurs more slowly in the kidney than animals challenged with yeast-phase (Table 1) in the heart during Candida infection. Although and filamentous-phase (Table 6) cells were com- large numbers of CFU of C. albicans were repared, the numbers of CFU were significantly covered from the kidney 12 h postchallenge, a lower in all organs of animals injected with fila- cellular infiltrate was not evident at that time. A kidney infiltrate has been observed by others mentous-phase cells. at 8 to 16 h, 24 h, 48 h, and 4 days or longer DISCUSSION postinfection (1, 12, 19, 21). The elevated BUN in animals challenged with The mortality observed after intravenous challenge of mice with C. albicans but not with C. albicans suggested decreased glomerular filC. guilliermondii is consistent with reports in tration; yet there was no histopathology evident the literature (1, 6, 9, 12, 13, 16, 21, 25, 28). The in the kidney 12 h postchallenge. This observamoribund appearance of animals 12 h postchal- tion requires additional comments. Although the lenge with C. albicans prompted examination of BUN of animals challenged with C. albicans the organ populations of the yeast at'this time, was elevated over that of controls, the serum and the highest numbers of CFU were recovered creatinine of the moribund animals was equivafrom heart and kidney homogenates. Quantita- lent to that of controls, suggesting that severe
996
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renal damage had not occurred. In addition, when total renal function was ablated by bilateral nephrectomy, the BUN of the mice when moribund exceeded 200 mg/100 ml (unpublished data), making it unlikely that the BUN observed in C. albicans-challenged mice was in the terminal stages of azotemia. Furthermore, it is known that in advanced heart failure decreased renal perfusion may result in subnormal glomer-
ular filtration which is evidenced in elevated BUN concentrations (4)-prerenal azotemia. In view of the observed cardiac histopathology and the absence of other indicators of severe renal damage, it is entirely possible that a condition of prerenal azotemia existed in those animals dying acutely from systemic C. albicans infection. Elevated serum levels of lactic dehydrogenase
SERUM BIOCHEMISTRY IN SYSTEMIC CANDIDIASIS
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TABLE 5. Effect of serum incubation upon the total mortality observed after intravenous challenge with 106 cells of the dimorphic phases of C. albicans Time of incubation" Percent mortality with inocula from Serum 37"C' Serum 25"C (min) 90 70 11
100' 100 100
30 60 180
"Sera were heat inactivated for 30 min at 56"C prior to inoculation with Candida. 'At 37"C, the percentage of cells that were filaments after 30, 60, and 180 min of incubation was (respectively): less than 10, greater than 60, greater than 90. 'Nine or 10 animals per experimental group. TABLE 6. Organ populations 12 h after intravenous challenge with 106 filamentous-phase C. albicans cells Organ
Brain Heart Kidney Liver Lung
Log1 CFU/g (wet wt) of organ ± 0.08a ± 0.24 ± 0.03 ± 0.12 ± 0.09 ± 0.07
4.18 4.57 5.21 4.67 4.91 4.33
Spleen Mean ± standard error. Each value represents three experiments, each comprising three animals. a
and serum glutamic oxalacetic and pyruvic transaminases were also documented 12 h postchallenge with C. albicans but not with C. guilliermondii. Although injury of either cardiac or hepatic tissue will elevate the serum levels of these enzymes, the absence of hepatic pathology in the animals examined here suggests that the increased serum concentration of these enzymes most likely reflects the cardiac pathology ensuing from lethal challenge with C. albicans. These biochemical alterations provide a quantitative measurement of events occurring during Candida infection. A number of years ago, endotoxin was postulated to account for Candida pathogenicity (38). More recently, phenol extraction of Candida cells resulted in a fraction which was lethal to mice upon injection (17, 18). However, the data in our report indicating that nonviable C. albicans cells were unable to produce either tissue pathology or elevated serum levels of BUN and creatine phosphokinase suggest that factors other than heat-stable cell wall components of the organism were responsible for these effects. Many investigators have observed the dimorphic phases of C. albicans in tissue (1, 6, 16, 20, 30, 42), and several investigators have compared the pathogenicity of the two phases as
997
well (8, 26, 40). Simonetti and Strippoli (40), for example, using the intraperitoneal route, reported that the mean time of death was longer when mice were challenged with the filamentous phase rather than the yeast phase. In the preparation of their inocula, however, filamentousphase cells were incubated in serum prior to injection whereas yeast-phase cells were not. Consequently, their experiments do not rule out the possibility that serum factors were responsible for the decreased virulence of filamentousphase cells. Recently, Mardon et al. (26) harvested yeast and filamentous phases of C. albicans from a synthetic medium after 3, 6, and 9 h of incubation and found greater mortality in mice challenged with the yeast phase. The inocula containing filamentous cells, however, i.e., those inocula harvested after 6 and 9 h of incubation, contained fewer viable units than corresponding yeast-phase inocula. Subsequently, Evans and Mardon (8) examined the fate of radioactively labeled cells during systemic infection and reported that yeast-phase cells multiplied significantly in the kidney 24 to 96 h after injection whereas filamentous-phase cells did not. Our data, obtained from experiments in which equivalent numbers of yeast and filamentous inocula prepared under identical cultural conditions were used, support those in the literature in that decreasing mortality was observed as more cells in the inoculum became filamentous. These experiments utilized cells at earlier stages of morphogenesis than those previously used (26). Although it cannot be ruled out that biochemical differences between the dimorphic phases of C. albicans may bring about their distinctly different pathological effects, the experiments in which C. albicans was quantitated from various organs suggest that concomitant host responses contribute to the less severe effects produced by filamentous-phase cells. After challenge with equal numbers of the two phases, the number of CFU in all organs was significantly lower when filamentous-phase cells were injected. A serum factor has been reported which clumps filamentous-phase C. albicans in vitro (5, 22). This factor may also act in vivo to effectively reduce the number of filamentous cells reaching the organs. These studies have demonstrated that characteristic serum biochemistries are found during the course of fatal Candida infection. Experiments are in progress to determine the factor(s) of C. albicans responsible for its pathological effects. ACKNOWLEDGMENTS This investigation was supported by General Research Support grant no. 17 from the University of Cincinnati.
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OBLACK, SCHWARZ, AND HOLDER
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IMMUNITY, Mar. 1978, p. 992-998
Vol. 19, No. 3
0019-9567/78/0019-0992$02.00/0 Copyright © 1978 American Society for Microbiology
Printed in U.S.A.
Biochemical Examination of Sera During Systemic Candida Infection in Mice DONNA OBLACK,1 JAN SCHWARZ,2 AND IAN A. HOLDER3* Departments of Microbiology' and Pathology, University of Cincinnati College of Medicine, and Shriners Burns Institute,3 Cincinnati, Ohio 45219 Received for publication 6 July 1977
Candida pathogenesis was examined by intravenous challenge of mice with either C. albicans or C. guilliermondii. Animals were moribund 12 h postchallenge with C. albicans and were found to have the greatest number of organisms in the heart and kidney, severe interstitial myocarditis, and elevated serum levels of blood urea nitrogen, creatine phosphokinase, serum glutamic oxalacetic transaminase, serum glutamic pyruvic transaminase, and lactic dehydrogenase. In contrast, challenge with C. guilliermondii resulted in a significantly lower renal census, no myocarditis, and no significant change in the concentration of these serum constituents. Challenge with nonviable C. albicans did not produce the effects observed with viable organisms. Moreover, challenge with filamentous C. albicans resulted in biochemical alterations of lower magnitude and in lower mortality rates. These results indicated that altered serum biochemistries were correlated with the histopathology of fatal Candida infection and that there were distinct differences with C. guilliermondii and the dimorphic phases of C. albicans.
genus Candida, one pathogenic and the other nonpathogenic, were used in these studies to enable recognition of those events that are unique to the pathogenic organism. Using the biochemical alterations described, we examined several variables for their effect on Candida pathogenicity. (These data were presented in part at the 76th Annual Meeting of the American Society for Microbiology, Atlantic City, N.J., 2-7 May
The frequency of deep fungal infection has increased steadily during the past 20 years, with yeasts of the genus Candida emerging as a very common cause of fungal infection of compromised hosts (2, 3, 11, 33, 37). Our knowledge of the events occurring during systemic Candida infection is mainly limited to descriptions of the host response against the yeast. For example, several investigators have examined the histopathology of systemic C. albicans infection. Postmortem examinations of human tissues have shown the kidney to be the organ most frequently containing lesions (15, 23, 30). In addition to renal involvement, at least 50% of patients were found to have lesions in the heart (15, 20, 23, 30, 37). Similarly, in murine models of C. albicans infection, the involvement of the kidney has been noted frequently (13, 16, 21, 28). Descriptions of gross and microscopic examination of this organ cite extensive abscess formation in which both yeast cells and pseudohyphae are present. In some cases (1, 6, 16, 19, 21, 42), the heart also has been involved with myocarditis and abscess formation. The heart has not been cited in all reports, however, so the frequency of its involvement may be underestimated. This study was undertaken to identify biochemical alterations which reflect the histopathology of experimental systemic C. albicans infection in mice. Two different species of the
1976.) MATERIALS AND METHODS Mice. Female, outbred Swiss mice, strain CD-1 (Charles River, Wilmington, Mass.), weighing 22 to 24 g were used in all experiments. Preparation of inocula. The two species of Candida utilized, C. albicans strain CA 2 and C. guilliermondii strain F 17, were obtained from the Mycology Laboratory of the Cincinnati General Hospital. Organisms were grown for 17 h at 370C in Sabouraud dextrose broth (SDB), harvested by centrifugation, and washed three times in sterile distilled water. Inocula for injection were adjusted to the standard concentration by use of a hemacytometer. Dilutions of inocula plated on Sabouraud dextrose agar (SDA) plates in triplicate showed that greater than 98% of the cells were viable. A 0.1-ml amount of the culture suspension was injected into a lateral tail vein. Animals were challenged in groups of 10. Nonviable cells were prepared by heating suspensions of C. albicans in a water bath at 560C for 90 to 120 min. Samples of the heat-treated suspensions were 992
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plated on SDA plates to confirm nonviability. Histology. At 12 h postinfection, organs were excised and fixed for 24 h in 10% buffered Formalin. Duplicate sections were prepared from each organ. One section was stained with hematoxylin-eosin (24), and the other was stained with methenamine-silver (10) with hematoxylin-eosin counterstain. Colony counts. The numbers of yeasts in various organs of infected animals were determined 12 h postchallenge by the method of Saymen et al. (39). In each experiment, the like organs from three animals were weighed and then homogenized as one sample. The number of yeasts in each homogenate was determined by dilution in SDB and plating on duplicate SDA plates. Plates were incubated at 37°C for 48 to 72 h. The total count in each organ was expressed as the number of colony-forming units (CFU) per gram (wet weight) of organ. Numbers of CFU are reported in log units as the mean of three experiments. Serum chemistries. Groups of animals were anesthetized with methoxyfluorane, and 100 to 150 ,tl of blood was obtained from the retro-orbital sinus 11 h prior to challenge with either organism; controls were injected with the diluent alone. A second blood sample was collected from each animal 11 to 12 h postinjection. A Clinicard 368 Analyzer (Instrumentation Laboratory, Inc., Lexington, Mass.) was used to analyze 25 ,ul of individual sera for the concentration of blood urea nitrogen (BUN) by the direct assay method of Crocker (7). From a control group and the two Candida-challenged groups of animals, other blood samples were obtained by cardiac puncture 12 h postinjection, and 100-,ul amounts of individual sera were analyzed for the concentration of creatine phosphokinase on a Clinicard 368 Analyzer. This methodology is based on the coupled enzymatic method of Oliver (29) with the modifications made by Rosalki (36) and Hess (14). Levels of other serum constituents were determined on pooled sera obtained from groups of 20 to 30 animals 12 h postchallenge with Candida. Sera from 20 uninfected mice were pooled and used as a control. Serum electrolytes (sodium, potassium, chloride) and carbon dioxide in the sera were determined by use of Stat/Ion Instrumentation (Technicon Corp., Tarrytown, N.Y.). Other serum constituents (total protein, albumin, calcium, phosphorus, glucose, uric acid, creatinine, total bilirubin, alkaline phosphatase, lactic dehydrogenase, and serum glutamic oxalacetic transaminase) were analyzed by use of a Sequential Multiple Analyzer-12 (Technicol Corp.). The concentration of serum glutamic pyruvic transaminase was determined by the change in ultraviolet absorbance at 340 nm with a Centrifichem System 400 (Union Carbide Corp., Rye, N.Y.). Statistical analysis. Student's t test was used to analyze the variance between experimental groups. Data were considered significant at a level of P < 0.025. Mortality studies of the dimorphic phases of C. albicans. Blastospores of C. albicans were harvested after 18 to 22 h of growth on SDA plates at 37°C and were inoculated into heat-inactivated normal human sera. Upon incubation at 37°C, these cells undergo transition to a filamentous phase (32, 41). In contrast,
993
similar cultures maintained at 250C retain the yeastphase morphology. Cells were collected after 30, 60, or 180 min of incubation at the two temperatures, washed, and injected intravenously into groups of 9 or 10 animals. The results are expressed as the percent mortality during the experimental period. The difference between experimental groups was analyzed with the chi-square test (31).
RESULTS Pathogenicity of Candida. Intravenous challenge of mice with 106 C. albicans cells resulted in a rapidly lethal infection. This dosage was completely lethal (LDioo) in 3 days. Within the first 24 h, 50% of the experimental group died, and the moribund appearance of mice 12 h postchallenge was most striking. In contrast, no deaths were recorded in 28 days when mice were challenged with equivalent numbers of C. guilliermondii cells. Colony counts. The organ populations of Candida 12 h postchallenge are presented in Table 1. The greatest numbers of CFU of C. albicans were found in the heart and kidney of the moribund animals. When animals challenged with the two species were compared, the numbers of CFU were equivalent in the brain, heart, liver, and lungs. Statistically significant greater numbers of C. albicans than C. guilliermondii were found in the kidney; in one organ, the spleen, C. guilliermondii was present in number one log higher than C. albicans. Histology. Histopathological examination of heart tissue 12 h postchallenge revealed severe focal myocarditis only in those animals challenged with C. albicans. Numerous foci of myocarditis showing microabscesses and tissue destruction were observed. Using the methenamine-silver stain, we observed yeast cells and pseudohyphae of C. albicans in these lesions. There was complete absence of inflammation in the myocardium of animals challenged with C. TABLE 1. Organ populations 12 h after intravenous challenge with Candida Logio CFU/g (wet wt) of organ Organ C. albicans
C. guilliernondii
4.12 ± 0.16 Brain .4.53 ± 0.04a 6.02 ± 0.14 Heart .6.06 ± 0.19 5.93 + 0.09 5.22 ± 0.09b Kidney .. 5.36 ± 0.08 Liver .5.52 ± 0.08 5.59 ± 0.09 Lung .5.61 ± 0.04 4.86 ± 0.11 5.83 + 0.25b Spleen aMean ± standard error. Each value represents three experiments, each comprising three animals. b p < 0.025, calculated from Student's t test comparing the groups challenged with C. albicans and C.
guilliermondii.
994
INFECT. IMMIJN.
OBLACK, SCHWARZ, AND HOLDER
Although large numbers of CFU of C. albihad also been recovered from kidney homogenates 12 h postinfection, a cellular infiltrate was not present in the kidney at this time. BUN. Abnormalities of kidney function were observed in mice 12 h postinfection with the yeast phase of C. albicans (Table 2). The BUN of animals challenged with C. albicans was significantly higher than that of uninfected controls. Challenge with an equivalent or 10-fold higher number of C. guilliermondii cells did not result in an elevation of the BUN. Challenge with 106 or 107 nonviable C. albicans cells was also without effect in this regard. BUN elevation was observed only when animals were challenged with yeast-phase cells of C. albicans; intravenous injection of equivalent numbers of filamentous cells did not produce a significant change. In two experiments, the serum BUN level after Candida challenge was significantly lower than that observed prior to challenge when both samples were collected from the same animal (Table 2, see BUN data with 106 C. guilliermondii and 106 nonviable C. albicans cells). Collecting two blood samples from uninfected control animals also resulted in a significantly lower BUN in the second sample. To avoid this artifact of significantly lowered BUN levels resulting from multiple bleedings, blood samples from uninfected and infected animals in the other experiments were collected from different animals. Creatine phosphokinase. Sera from animals challenged with both Candida species were analyzed for the concentration of creatine phosphokinase, an enzyme localized in heart, skeletal muscle, and brain (34) which is released in quantity into the circulation after cellular destruction
in those tissues. The data presented in Table 3 indicate that acute injury of myocardial tissue, consistent with the histopathological evidence, occurs during systemic infection with C. albicans. The serum creatine phosphokinase level of mice, 12 h postchallenge, was elevated 30-fold over controls. That of mice challenged with a comparable or 10-fold higher number of C. guilliermondii cells was not significantly different from control animals. Elevated serum creatine phosphokinase levels were not observed when animals were challenged with nonviable C. albicans, either at the same dosage or at a 10-fold greater dosage. Furthermore, nonviable cells did not elicit a cellular infiltrate in the myocardium of these animals. When animals were challenged with filamentous C. albicans, creatine phosphokinase was elevated to an intermediate level (about fourfold, Table 3). Challenge with 106 or 107 nonviable filamentous cells did not result in significantly elevated creatine phosphokinase levels. Electrolytes and other serum chemistries. To document other biochemical alterations during Candida infection, pooled sera from uninfected controls and from animals challenged with either C. albicans or C. guilliermondii were assayed for the concentration of 16 additional components. The serum concentrations of 13 of the 16 additional components were equivalent in the three groups of animals. However, the concentrations of three enzymes in the sera of animals challenged with C. albicans were found to be elevated twofold or greater over that of uninfected controls (Table 4). At 12 h after challenge, there was a fivefold increase in lactic dehydrogenase, an eightfold increase in serum glutamic oxalacetic transaminase, and a threefold increase in serum glutamic pyruvic transaminase. Elevated serum concentrations of these enzymes were not observed in animals after challenge with C. guilliermondii.
TABLE 2. Evaluation of kidney function 12 h after intravenous challenge with Candida
TABiF, 3. Evaluation of cardiac injury 12 h after intravenous challenge with Candida Creatine phosphokinase (IU/liter)
guilliermondii despite the presence of equivalent numbers of organisms documented in Table 1. cans
BUN (mg/100 ml)
Challenge
Challenge
Uninfected
Candida chal-
controls
lenged
C. albicans cells 106 cells, nonviable l07 cells, nonviable
106
106 cells, filamentous
29.2 + 1.4 (1O)" 32.5 + 1.0 (10) 24.2 1.2 (10) 28.5 1.5 (10)
51.9 26.2 24.8 27.8
22.7 28.2
± ± ± ±
5.4b (10) 1.2 1.0 0.8
(10) (10) (10)
C. guilliermondii
106 cells 107 cells
29.7
±
1.0 (15)
27.6
±
1.5 (10)
± ±
1.9 (9) 1.7 (9)
aMean ± standard error. The numbers in parentheses represent total animals tested; each animal was assayed once. b < 0.025, calculated from Student's t test comparing uninfected controls and Candida-challenged groups.
Uninfected controls
Candida challenged
C. albicans 10" cells 248 ± 40 (8)" 7,908 ± 1,002" (10) 10" cells, nonviable 182 ± 19 (10) 193 ± 21 (10) 107 cells, nonviable 285 ± 23 (8) 331 ± 34 (7) ± 10" cells, filamen- 285 27 (9) 1,069 ± 81" (10) tous C. guilliermondii 10' cells 395 + 80 (10) 292 ± 59 (10) 10' cells 205 ± 31 (9) 346 ± 66 (10) " Mean ± standard error. The numbers in parentheses represent total animals tested; each animal was assayed once. P < 0.025, calculated from Student's t test comparing uninfected controls and Candida-challenged groups.
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TABLE 4. Serum enzyme levels 12 h after intravenous challenge with 10U Candida cells Enzyme
Uninfected controls
C. albicans challenged
C. guihallerinond
challenged
560 522-600a 2,400 Lactic dehydrogenase .... 135 115-124 1,000 Glutamic oxalacetic transaminase 20 49 18 Glutamic pyruvic transaminase " Enzyme concentration in milliunits per milliliter. Each value given represents assay of pooled sera collected from 20 to 30 animals.
Virulence of the dimorphic phases of C. tive examinations of Candida growth in vivo albicans. The previous data, indicating that have been only infrequently reported. Kemp and yeast-phase cells produced greater elevations of Solotorovsky (19) investigated Candida infeccreatine phosphokinase and BUN than filamen- tion in mice and reported increasing CFU in the tous cells, prompted investigation of the differ- heart and kidney during the early stages of the infection. Louria et al. (21) found high numbers ences in virulence between the two phases. Filamentation of C. albicans in serum is a of CFU of C. albicans in the kidneys of mice species-, substrate-, time- and temperature-de- when moribund within 6 days after challenge. It pendent phenomenon (41). Figure 1 depicts the has recently been reported that, on a weight progression of filament formation by C. albicans basis, the kidney clears a greater percentage of upon incubation in serum for various times at the Candida inoculum than the liver, lungs, or 370C. At the time of inoculation, only yeast- spleen (35). Whether this capacity contributes phase cells were present (Fig. 1A). However, to the ultimate growth of C. albicans in the within 30 min of incubation, an occasional yeast kidney remains to be determined. One unexcell exhibited the beginning of the filament (Fig. pected finding of the quantitation experiments 1B). After 1 h of incubation more than two- was the observation of 10-fold greater CFU of C. thirds of the yeast cells had formed short fila- guilliermondii than of C. albicans in spleen ments (Fig. 1C), and after 3 h of incubation homogenates 12 h postchallenge. Inasmuch as greater than 90% of the yeast cells had filaments neither Candida species multiplies in the spleen (Fig. 1D), most of which were longer than those (9, 19, 27, 35), this observation may have little observed at 1 h. Yeast cells inoculated into se- bearing on Candida pathogenesis. Although myocarditis has not been regularly rum and maintained at 250C for comparable emphasized in Candida infection, our observatimes retained a yeast morphology. Presented in Table 5 are the mortalities ob- tion of myocarditis occurring acutely in the served after intravenous challenge with 106 cells course of fatal Candida infection supports the of the dimorphic phases of C. albicans incubated work of several investigators (1, 6, 16, 21). This for 30, 60, or 180 min in serum. As the transition response was specifically produced by C. albifrom the yeast to the filamentous phase pro- cans, for, despite the presence of equivalent numbers of CFU in the myocardium 12 h postceeded, a decrease in mortality occurred. The populations of C. albicans in various challenge, a cellular infiltrate was not elicited by organs 12 h after challenge with 106 filamentous C. guilliermondii. Histological examination of cells are presented in Table 6. The greatest kidney sections indicated that a cellular host number of CFU were found in the kidney. When response occurs more slowly in the kidney than animals challenged with yeast-phase (Table 1) in the heart during Candida infection. Although and filamentous-phase (Table 6) cells were com- large numbers of CFU of C. albicans were repared, the numbers of CFU were significantly covered from the kidney 12 h postchallenge, a lower in all organs of animals injected with fila- cellular infiltrate was not evident at that time. A kidney infiltrate has been observed by others mentous-phase cells. at 8 to 16 h, 24 h, 48 h, and 4 days or longer DISCUSSION postinfection (1, 12, 19, 21). The elevated BUN in animals challenged with The mortality observed after intravenous challenge of mice with C. albicans but not with C. albicans suggested decreased glomerular filC. guilliermondii is consistent with reports in tration; yet there was no histopathology evident the literature (1, 6, 9, 12, 13, 16, 21, 25, 28). The in the kidney 12 h postchallenge. This observamoribund appearance of animals 12 h postchal- tion requires additional comments. Although the lenge with C. albicans prompted examination of BUN of animals challenged with C. albicans the organ populations of the yeast at'this time, was elevated over that of controls, the serum and the highest numbers of CFU were recovered creatinine of the moribund animals was equivafrom heart and kidney homogenates. Quantita- lent to that of controls, suggesting that severe
996
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FIG. 1. Progression of in vitro filament formation by C. albicans upon incubation in serum at 37"C for (A) 0 min, (B) 30 min, (C) 60 min, and (D) 180 min. Wet mount. xl,008.
renal damage had not occurred. In addition, when total renal function was ablated by bilateral nephrectomy, the BUN of the mice when moribund exceeded 200 mg/100 ml (unpublished data), making it unlikely that the BUN observed in C. albicans-challenged mice was in the terminal stages of azotemia. Furthermore, it is known that in advanced heart failure decreased renal perfusion may result in subnormal glomer-
ular filtration which is evidenced in elevated BUN concentrations (4)-prerenal azotemia. In view of the observed cardiac histopathology and the absence of other indicators of severe renal damage, it is entirely possible that a condition of prerenal azotemia existed in those animals dying acutely from systemic C. albicans infection. Elevated serum levels of lactic dehydrogenase
SERUM BIOCHEMISTRY IN SYSTEMIC CANDIDIASIS
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TABLE 5. Effect of serum incubation upon the total mortality observed after intravenous challenge with 106 cells of the dimorphic phases of C. albicans Time of incubation" Percent mortality with inocula from Serum 37"C' Serum 25"C (min) 90 70 11
100' 100 100
30 60 180
"Sera were heat inactivated for 30 min at 56"C prior to inoculation with Candida. 'At 37"C, the percentage of cells that were filaments after 30, 60, and 180 min of incubation was (respectively): less than 10, greater than 60, greater than 90. 'Nine or 10 animals per experimental group. TABLE 6. Organ populations 12 h after intravenous challenge with 106 filamentous-phase C. albicans cells Organ
Brain Heart Kidney Liver Lung
Log1 CFU/g (wet wt) of organ ± 0.08a ± 0.24 ± 0.03 ± 0.12 ± 0.09 ± 0.07
4.18 4.57 5.21 4.67 4.91 4.33
Spleen Mean ± standard error. Each value represents three experiments, each comprising three animals. a
and serum glutamic oxalacetic and pyruvic transaminases were also documented 12 h postchallenge with C. albicans but not with C. guilliermondii. Although injury of either cardiac or hepatic tissue will elevate the serum levels of these enzymes, the absence of hepatic pathology in the animals examined here suggests that the increased serum concentration of these enzymes most likely reflects the cardiac pathology ensuing from lethal challenge with C. albicans. These biochemical alterations provide a quantitative measurement of events occurring during Candida infection. A number of years ago, endotoxin was postulated to account for Candida pathogenicity (38). More recently, phenol extraction of Candida cells resulted in a fraction which was lethal to mice upon injection (17, 18). However, the data in our report indicating that nonviable C. albicans cells were unable to produce either tissue pathology or elevated serum levels of BUN and creatine phosphokinase suggest that factors other than heat-stable cell wall components of the organism were responsible for these effects. Many investigators have observed the dimorphic phases of C. albicans in tissue (1, 6, 16, 20, 30, 42), and several investigators have compared the pathogenicity of the two phases as
997
well (8, 26, 40). Simonetti and Strippoli (40), for example, using the intraperitoneal route, reported that the mean time of death was longer when mice were challenged with the filamentous phase rather than the yeast phase. In the preparation of their inocula, however, filamentousphase cells were incubated in serum prior to injection whereas yeast-phase cells were not. Consequently, their experiments do not rule out the possibility that serum factors were responsible for the decreased virulence of filamentousphase cells. Recently, Mardon et al. (26) harvested yeast and filamentous phases of C. albicans from a synthetic medium after 3, 6, and 9 h of incubation and found greater mortality in mice challenged with the yeast phase. The inocula containing filamentous cells, however, i.e., those inocula harvested after 6 and 9 h of incubation, contained fewer viable units than corresponding yeast-phase inocula. Subsequently, Evans and Mardon (8) examined the fate of radioactively labeled cells during systemic infection and reported that yeast-phase cells multiplied significantly in the kidney 24 to 96 h after injection whereas filamentous-phase cells did not. Our data, obtained from experiments in which equivalent numbers of yeast and filamentous inocula prepared under identical cultural conditions were used, support those in the literature in that decreasing mortality was observed as more cells in the inoculum became filamentous. These experiments utilized cells at earlier stages of morphogenesis than those previously used (26). Although it cannot be ruled out that biochemical differences between the dimorphic phases of C. albicans may bring about their distinctly different pathological effects, the experiments in which C. albicans was quantitated from various organs suggest that concomitant host responses contribute to the less severe effects produced by filamentous-phase cells. After challenge with equal numbers of the two phases, the number of CFU in all organs was significantly lower when filamentous-phase cells were injected. A serum factor has been reported which clumps filamentous-phase C. albicans in vitro (5, 22). This factor may also act in vivo to effectively reduce the number of filamentous cells reaching the organs. These studies have demonstrated that characteristic serum biochemistries are found during the course of fatal Candida infection. Experiments are in progress to determine the factor(s) of C. albicans responsible for its pathological effects. ACKNOWLEDGMENTS This investigation was supported by General Research Support grant no. 17 from the University of Cincinnati.
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OBLACK, SCHWARZ, AND HOLDER
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