MYCOSES 35,
83-88 (1992)
ACCEPTED: FEBRUARY 29, 1992
A modified method for experimental candidosis in mice avoiding lethality Eine modifizierte Methode der nicht-letalen, experimentellen Mause- Candidose H. Hanel, W. Raether, M. Uphoff, Bettina Braun, J. Kalisch and Simone Pastowsky Key words. Candida albicans, infection model, non-lethal, mice, screening. Schlusselworter. Candida albicans, Infektionsmodell, nicht letales, Maus, Screening.
Summary. A model is presented which selected one out of 150 Candida albicans strains for the evaluation of antifungal agents. The mice were inoculated with 6 x lo5 CFU of strain 352 into the tail vein. The strain has a moderate phospholipase B (PLB) activity in uilro and was originally isolated from a stool sample from a patient in an intensive care unit. This infection leads to very little suffering in the infected animals during the 6-day observation period. Kidney counts at day 5 after infection can give a first indication for a possible fungistatic mechanism. Possible interesting drugs can then be evaluated by a second set of experiments using a longer observation time to investigate the compounds for fungicidal properties. The model suggests that screening for systemic antifungals by avoiding lethality of mice in the first place can be done. Zusammenfassung. Tiefe Candidosen gehoren zu den Krankheitsbildern mit Todesfolge, die in den letzten Jahren besonders stark zugenommen haben. Eine unzureichende Diagnostik und Probleme bei der Therapie haben dazu gefuhrt, dafi immer mehr Patienten mit gestorter zellularer Abwehr bei der Obduktion Candida albicansHerde aufweisen. Trotz guter Erfolge in der Therapie durch die neuen Triazole bleibt das Medikament der ersten Wahl Amphotericin B. Dieses sehr unvertragliche Praparat darf nur u n ter strenger Risikoabwagung verabreicht werden. Hoechst AG, Frankfurt/Main-Hochst, Germany. Correspondence: Dr Heinz Hanel, Hoechst AG, P.O. Box 800320, D-W-6230 Frankfurt/Main 80, Germany.
Es besteht demzufolge ein dringender Bedarf an einer Entwicklung vertraglicher und fungizider Arzneimittel fur tiefe Candidosen. Bisher wurden die Testsubstanzen im letalen Mausemodell gepriift. Hierbei kam es etwa 2-5 Tage nach intravenoser Infektion mit den Hefepilzen zum Tod der Tiere durch Nierenversagen. Die Versuchstiere hatten insbesondere wahrend der letzten 2 Lebenstage eine hohe Belastung durch Schmerzen und Leiden. Dies zeigte sich am gestraubten Fell und a n Apathie. Wir stellen hier ein Model1 vor, in dem ein Candida albicans-Stamm ausgewahlt wurde, der bei den Tieren weder zu erkennbaren Verhaltensauffalligkeiten, noch zu dem sonst aufgetretenen gestraubten Fell fuhrt. Trotzdem zeigen die durchgefuhrten Therapieversuche mit dem neuen Pilzstamm, daR das Ansprechen auf eine breite Palette von Handelszubereitungen genau dem klinischen Bild entspricht. Die Methode eignet sich somit, klinisch relevant verschiedene Verbindungen auf ihren systemischen Effekt gegeniiber tiefen Candidosen zu prufen, ohne die Versuchstiere dem bisherigen Leidensdruck aussetzen zu mussen.
Introduction Murine models for the evaluation of antifungal agents are generally accepted. Fatal infections with mice can readily be produced by injecting between lo5 and lo7 viable yeast cells per mouse intravenously (for an extensive review see Ref. 1). However, we have some doubts about using an inoculum which causes death in sometimes
84
H. HANELET AL.
less than 4 days. With low kidney counts [2] high inocula may lead to an obstructive mechanism with misleading consequences for the evaluation of antimycotic agents. It has been suggested that one should wait for as long as 70 days to consider a drug as active in terms of preventing lethality [3]. In accordance with Ryley [4], we suggest that lower inocula with less pathogenic strains .are helpful to evaluate therapeutic properties of the used antifungals rather then just cause slightly extended survival rates. Additionally the German “Tierschutzgesetz” (law of animal protection) asks for evaluation methods which cause as little suffering and irritation of the experimental animals as possible. We therefore present the results from studies of a large selection of Candida albicans strains which were investigated for their ability to cause persistent kidney infections without obvious signs of fatigue in the mice. Fluconazole, itraconazole, ketoconazole and amphotericin B were used in this model to evaluate the comparability with the lethal types of infections reported previously [5, 61.
obtained from Sigma Corp. (Munchen). I traconazole was a generous gift from Janssen and fluconazole was kindly supplied by Pfizer. In vivo evaluation All strains were tested using four different inocula: 1 x lo5, 3 x lo5, 6 x lo5 and 1 x lo6 CFU/animal given in 0.2 ml of physiological NaCl solution intravenously into the tail vein. Mortality and appearance of the mice was recorded daily for 21 days. T h e treatment scheme of animals for the various antimycotics is indicated in Table 5. Treatment was done twice daily beginning with D- 1 to D + 2 (D 3 respectively). Infection controls were treated with the solvent accordingly. At each day beginning one day after infection two animals from each group, treated and untreated, were killed, the kidneys dissected, weighed, homogenized and plated as described below. Kidneys from the treated group were taken, starting with D + 3 (Table 3).
+
Homogenization of kidneys and evaluation of CFU
Materials and methods Candida albicans strains Four strains of Candida albicans were selected from 150 strains obtained by a centre for laboratory medicine in Germany. The isolates were cultured from stool samples. I n previous experiments we investigated a number of parameters including mortality of mice, phospholipase production, and left and right kidney counts after intravenous infection among others [7]. One C. albicans strain (1 75) had been our internal standard for intravenous infection; it was obtained 15 years ago from Hoffmann La Roche, and was originally isolated from a patient with a systemic candidosis. All selected strains (Nos 352, 371, 410 and 420) belong to serotype A (for a discussion of the different infectivities between serotypes A and B see Ref. 8).In vitro evaluation of the phospholipase activity was done with the agar-plate method described by Price el al. [9]. The phospholipase activity of all strains was compared using strain 175 as a standard (Table 1). With this method we can evaluate phospholipase B (PLB) (and most likely the lysophospholipases) as has been published [ lo].
Compounds Amphotericin B was used in its commercial formulation Amphotericin B@and ketoconazole was
Both kidneys of each mouse were homogenized in 10 ml of physiological NaCl solution using an Ultra Turrax@ (Buhler, Germany) blender at 20,000 rpm for 1 min. The undiluted mixture was then plated on malt-agar plates containing 1% streptomycin and 1% penicillin using a Spiral Plate@ (Spiral Systems Inc., USA) which applies 92 p1 onto the plates. After incubation of the plates at 36°C for 2 days, colonies were counted according to the evaluation scheme for the Spiral Plater@.
Additional tests with strain 352 Compounds tested with strain 352 were fluconazole, ketoconazole, itraconazole (all orally) and amphotericin B (i.p.) each with 2.5 and 1.25 mg kg-’ eight times. In a second set of experiments fluconazole 6 x (2.5 mg kg-’) was compared with fluconazole 8 x (2.5 mg kg-’) using strain 352.
Results Table 1 indicates a comparison between the in vilro detected phospholipase activity and the mean survival time of mice after challenge with the five selected strains. I t is evident that among these strains there is no linear relationship between PLB activity and speed of lethality. mycoses 35, 83-88 (1992)
EXPERIMENTAL CANDIDOSIS AVOIDING
and lethality in mice after i.v. injection of C. ulbicunr suspensions (strain 175: 6 x lo5 CFU/animal) Phospholipase
B activity (Yo)
175 352 37 1 410 420
100 66 111 96 40
+
Mean survival time (days) (10 mice each) RfSD 2.2 f0.6 6.5 f2.0 5.0 f 1.8 5.0f3.1 7.0f2.0
Strain 352, having a relatively low PLB production, led to a longer survival time than strain 371 which had shown the highest PLB activity among the five isolates. Table 2 indicates the CFU counts within the homogenized kidneys of the infected mice. All animals received placebo treatment only. Strain 175 produced lower counts after the highest (1 x lo6 CFU) inoculation compared with 3 x lo5 and 6 x lo5, especially at D + 4. CFU after 1 x lo5 were minimal. Strain 352 led to small CFU counts after 1 x lo5 and 3 x lo5 but high counts were achieved after 6 x lo5 and 1 x lo6. Strain 371 behaved similarly to 352 with lower counts after 1 x lo5. Strains 410 and 420 produced only rather limited counts irrespective of the inoculum used.
Table 2. CFU of Cundida albicuns in kidneys of mice infected with five different strains from 1 to
85
Table 3 gives the data from the fluconazoletreated groups. Most fluconazole-treated mice had dramatically reduced CFU values in the kidneys due to the treatment. Strain 175 was generally reduced from D + 3 to D 4 by 99% or more. At D + 5 , the death of the infection control animals meant that the reduction could not be evaluated. Strains 352 and 371 showed a decreasing reduction a t D + 4 and D + 5 after termination of the treatment. Strain 410 caused higher CFU values than strain 420. Correspondingly, the reduction of 410 was generally between 87 and 98%) irrespective of time and infection dose. Strain 420 showed varying reductions with a peak at D + 5 . A summary of the data for illness is shown in Table 4. Strain 175 led to a distinctive mortality beginning as early as D+2. Mice infected with strain 175 showed the highest scores of illness throughout the experiment. Strains 352 and 371 led to single mortalities whereas strains 410 and 420 did not kill any mice within the 6-day scoring time. The score of illness increased with the inoculum except for strain 420 where mice appeared more ill after 3 x 1O5 than after 6 x 1O5 CFU/mouse. Taking all parameters together (CFU/mouse, score of illness, PLB production and therapeutic experiments) we selected strain 352 for further use in a mouse model, generally applying 6 x lo5 CFU by the intravenous route. Treatment tests with strain 352 are shown in
Table 1. Evaluation of phospholipase B activity in uifro
Strain no.
LETHALITY
to
5 days after infection (D + 1
D+5)
Strain no. 175
352
37 1
410
420
Inoculum/ mouse 1 x 105 3~ 105 6 x lo5 1 x 106 1 x 105 3 x 105 6 x lo5 1 x 106 1 x 105 3~ 105 6 x lo5 1 x lo6 1 x 105 3 x 105 6 x lo5 1 x 106 1 x lo5 3~ 1 0 5 6 x lo5 1 x lo6
mycoses 35, 83-88 (1992)
Observed CFU x lo5/" kidneys
D+ 1
D+2
D+3
D+4
D+5
0.03 0.4 0.1 1 4.1 1 0.06 0.27 0.76 1.11 0.12 0.33 0.23 0.35 0.008 0.052 0.036 0.48 0.012 0.05 0.027 0.19
0.36 0.93 3.76 6.44 0.39 1.47 36.6 70.0 0.23 0.49 70.0 70.0 0.081 0.49 0.78 1.5 0.095 0.03 0.046 1.9
0.074 5.1 12.0 12.2 0.7 1.3 70.0 38.5 0.06 35.3 70.0 0.6 1 0.42 2.91 0.6 I .46 0.06 0.002 0.002 4.07
0.64 39.0 38.0 0.15 0.90 1.62 4.78 43.8 0.36
0.22 1.85 dead dead 0.26 7.7 10.4 39.2 0.23 0.14 0.59 35.6 3.6 8.5 25.2 I .48 0.03 0.02 0.012 35.8
0.27
35.5 35.3 1.44 2.37 1.94 1.3 0.19 0.022 0.012 0.56
86
H.
H A N E L ET AL.
Table 3. CFU of Cundidu nlbicuns in kidneys of mice treated orally eight times with fluconazole 2.5 mg kg-’ after infection with five different strains from four different inocula Strain no.
175
352
37 1
410
420
D+3
Inoculum/ mouse
Ix 3~ 6x 1x Ix 3~
105 105 106
105 105 6 x lo5 1 x 106 I x 105 3~ 105 6 x lo5 1 x 106 I x 105
3~ 6x 1x Ix 3~ 6x 1x
CFU
(YO)
CFU
0.0
100 99.76 99.79 99.48 98.71 99.04 99.98 99.96 89.66 99.96 99.96 91.19 89.66 98.69 93.65 96.38 95.0 50.0 50.0 99.88
0.04 0.19 0.18 2.97 0.11 1.61 2.65 1.31 0.09 0.18 0.57 0.72 0.38 1.24 1.81 0.83 0.0 0.04
0.12 0.25 0.64 0.09 0.12 0.15 0.16 0.06 0.15 0.25 0.54 0.17 0.38 0.38 0.53 0.03 0.01 0.01 0.05
lo5
105 lo5 lo6 105 105 lo5 lo6
D+4
0.0 1 0.02
I
D+5 (YO)
CFU
(%I
99.37 99.95 99.95 0.0 98.78 90.06 94.46 99.70 97.52 93.38 99.84 99.80 97.36 94.76 90.66 93.59 100 81.82 91.67 99.64
0.28 2.1 1 2.15 1.32 1.26 1.77 1.42 4.07 0.17 0.15 0.32 0.76 1.48 1.18 5.08 1.89 0.0 0.01 0.01 0. I
87.27 88.56 r.d. r.d. 51.54 97.70 98.63 98.96 92.70 89.05 94.58 99.79 95.89 98.61 97.98 87.23 I00 99.47 91.67 99.97 I
CFU= CFU x 104/2kidneys;
(Yo)= Yo inhibition;
r.d. =respective control dead.
Table 4. Score values of ‘illness’ in mice infected intravenously with five different Cundidu ufbicuns strains one to five days after infection Strain no.
Inoculum
D+1
D+2
D+3 D+4
D+5
Table 5. It is obvious that all of the antimycotics used are effective to a varying degree, with amphotericin B (ip.) being the most effective,
followed by fluconazole, itraconazole and ketoconazole (orally).
Discussion Intravenous inoculation of mice with Candida albicans leads to graded fungal kidney counts. As Ryley et al. [3] demonstrated, high inocula (3 x lo6) lead to early death with low counts, whereas moderate inocula (3 x lo5) produced high counts in infections which some mice survived. The comparison of various C. albicans strains in our infection model reflects the need for a predictable method which avoids the early death due to kidney failure. Strain-dependent differences in susceptibility of mice were investigated by Marquis et al. [ 111. The authors mainly focused on the kidney weight as a parameter of infection. We used the fungal count only, for we could not see a definite correlation between the weight of a n infected kidney and the number of colony-forming units ([7] and unpublished results). This is in accordance with the histopathological investigations by Blyth [ 121 and Ryley & Ryley [13], both of whom demonstrated the different cellular reaction of kidneys towards blastoconidia and filaments which then lead to different kidney weights. Comparisons of different strains (wild types and mutants) have been made mycoses 35, 83-88 (1992)
EXPERIMENTAL CANDIDOSIS
87
AVOIDING LETHALITY
Table 5. Influence of treatment regimen on the number of CFU/kidney at D + 3 after Candida albicans intravenous infection of mice with strain 352 (6 x lo5 CFU/mouse)
Compound
Applications
Dose
(mg kg-’) Fluconazole (oral) Ketoconazole (oral) I traconazole (oral) Amphotericin B (i.P.) Infection control
1.25 2.5 2.5 1.25 2.5 1.25 2.5 1.25 2.5
8 6 8 8 8
a
8 8 8 8
but not in view of a decreased suffering [14]. The five strains investigated in our experiments differed in their ability to produce high kidney counts. As can be seen from the phospholipase comparison (Table 1) this cannot be the only factor determining pathogenicity. Besides phospholipase a number of other pathomechanisms have been discussed (for a recent review see Refs 15 and 16). As early as 1940, Henrici [I71 postulated an exotoxin produced by Candida albicans to explain effects he observed in experimental infections. Irrespective of the unresolved problems concerning the pathomechanism, we focused on the behaviour of the infected mice in order to prevent suffering of the animals. Table 4 indicates that our previous reference strain 175 led to early symptoms which were reflected by a wet appearance of the fur and loss of appetite. Strain 410 showed the least severe symptoms but failed to produce constant and reliable kidney counts. It is interesting to note that the CFU reduction due to fluconazole at D 5 was stronger than for the more aggressive strain 175, probably due to a slower tissue invasion and a different morphological appearance. It is generally accepted that the fungistatic azole compounds inhibit mycelia at a lower concentration than they do blastoconidia. We therefore selected strain 352 with a relatively low phospholipase activity for further evaluation in therapeutic experiments. We did not try to modulate the response of the host with antibiotics [18] or silica to modify the macrophage response [ 191 because a routine model for the evaluation of antifungal agents has to reflect the clinical situation as closely as possible. This is in accordance with the experiments of Hare & Loebenberg [20] who also used kidney counts for distinguishing between fungicidal and fungistatic compounds. Our results with fluconazole with the five strains show that the recent
+
mycoses 35, 83-88 (1992)
Mean CFU/kidney
Reduction
Mice
( x 103)
(Yo)
(n)
8.10 6.93 2.26 1512.12 330.82 115.16 26.23 1.61 0.48 1663.95
99.5 99.6 99.9 9.0 80.1 93.1 98.4 99.9 99.97
5 10
0.0
10
5 5 5 5 5 5 10
triazoles can reduce the colony-forming units dramatically under the experimental conditions tested. However, as Hare & Loebenberg [20] indicated, we observed high mortality after the termination of treatment with triazoles. Amphotericin B, on the other hand, obviously acts by the fungicidal way under these experimental conditions. We therefore suggest the screening of potential antifungal compounds with the short model first, evaluating the fungistatic properties. Suitable drugs should then be tested again by observing the treated mice for at least two months to investigate the fungicidal properties of the selected compounds. The development of an infection model in which the mice suffer to only a limited degree is necessary for the ethical justification for undertaking animal experiments in the routine search for new antifungal agents.
References 1 Miyaji, M. (1987) Animal Models in Medical Mycology. Boca Raton, Florida: C R C Press. 2 Ryley, J. F., McGregor, S., Lister, S. C. & Jackson, K. P. (1988) Kidney function in experimental systemic candidosis of mice. Mycoses 31, 203-207. 3 Ryley, J. F., McGregor, S. & Wilson, R. G. (1988) The activity of ICI 195739, a novel orally active bis-triazole, in rodent models of fungal and protozoal infections. Ann. N.Y. Acad. Sci. 544, 310-328. 4 Ryley, J. F. (1990) Screening and evaluation in vivo. Handbook of Experimental Pharmacology 96, 1 29- 147. 5 Troke, P. F., Andrews, R. J., Brammer, K. W., Marriott, M. S. & Richardson, K. (1985) Efficacy of UK-49,858 (Fluconazole) against Candida albicans experimental infections in mice. Antimicrob. Agents Chemother. 28, 8 I 5-8 18. 6 Ryley, J. F., & McGregor, S. (1988) A multi infection model for antifungal screening in vivo. 3. Antimicrob. Chemother. 22, 353-358. 7 Hanel, H., Schiitz, K. D., Buslau, M. & Holzmann, H. (1 990) Untersuchungen von Candida albicans-Isolaten aus Stuhlproben von Neurodermitikern. 24th Scientific Confer-
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8
9
10
11
12 13
H. HANELET AL.
me of the German-Speaking Mycological Society, Gottingen, Abstract No 24, p. 9. Polak, A., Odds, F. C., Liidin, E. & Scholer, H. J. (1985) Correlation of susceptibility test results in vitro with response in vivo: Ketoconazole therapy in a systemic candidiasis model. Chemotherapy 31, 395-404. Price, M. F., Wilkinson, I. D. & Gentry, L. 0. (1982) Plate method for detection of phospholipase activity in Candida albicans. Sabouraudia 20, 7- 14. Hanel, H., Menzel, I., Buslau, M. & Holzmann, H. (1989) Messung der Phospholipaseaktivitat von Cundidu albicans im Darm. In: Miiller, J., Ottenjann, R. & Seifert, J. (eds) Okosystem Dorm. Berlin: Springer, pp. 185-187. Marquis, G., Montplaisir, S., Pelletier, M., Mousseau, S. & Auger, P. (1986) Strain-dependent differences in susceptibility ofmice to experimental candidosis. 3.Infect. Dis. 151, 906-909. Blyth, W. (1959) Hostlparasite relationships in experimental moniliasis. Mycopath. Mycologia Applicata 10, 269-282. Ryley, J. F. & Ryley, N. G. (1990) Cundida albicansdo mycelia matter? Sabouraudia 28, 225-239.
14 Fromtling, R. A., Abruzzo, G. K., Edison, A. & Manning-Zweerink, W. (1988) Renal pathology and spleen cell chemiluminescence of mice infected with a wild-type and a low-virulence mutant of Candidu albicans. Zbl. Bakt. Hyg. A 268,405-415. 15 Ghannoum, M. A. & Abu-Elteen, K. H. (1990) Pathogenicity determinants of Candida. Mycoses 33, 265-282. 16 Odds, F. C. (1988) Candida and Candidosis. 2nd edn. London: Baillitre Tindall. 17 Henrici, A. T. (1940) 3. Bact. 39, 113-citated after Winner, H. I. (1958) An experimental approach to the study of infections by yeast-like organisms. Proc. Royal SOC.Med. 51, 496-499. 18 Blyth, W. (1958) The influence of antibiotics on experimental moniliasis. Mycopath. Mycologia Applicata 10, 91-1 12. 19 Sinha, B. K., Monga, D. P. & Prasad, S. (1987) Studies on the role of macrophages in experimental candidosis in mice. Mycoscs 30, 105-1 12. 20 Hare, R. S. & Loebenberg, D. (1988) Animal models in the search for antifungal agents. Am. SOC.Microbiol. NCWS 54, 235-239.
mycoses 35, 83-88 (1992)
83-88 (1992)
ACCEPTED: FEBRUARY 29, 1992
A modified method for experimental candidosis in mice avoiding lethality Eine modifizierte Methode der nicht-letalen, experimentellen Mause- Candidose H. Hanel, W. Raether, M. Uphoff, Bettina Braun, J. Kalisch and Simone Pastowsky Key words. Candida albicans, infection model, non-lethal, mice, screening. Schlusselworter. Candida albicans, Infektionsmodell, nicht letales, Maus, Screening.
Summary. A model is presented which selected one out of 150 Candida albicans strains for the evaluation of antifungal agents. The mice were inoculated with 6 x lo5 CFU of strain 352 into the tail vein. The strain has a moderate phospholipase B (PLB) activity in uilro and was originally isolated from a stool sample from a patient in an intensive care unit. This infection leads to very little suffering in the infected animals during the 6-day observation period. Kidney counts at day 5 after infection can give a first indication for a possible fungistatic mechanism. Possible interesting drugs can then be evaluated by a second set of experiments using a longer observation time to investigate the compounds for fungicidal properties. The model suggests that screening for systemic antifungals by avoiding lethality of mice in the first place can be done. Zusammenfassung. Tiefe Candidosen gehoren zu den Krankheitsbildern mit Todesfolge, die in den letzten Jahren besonders stark zugenommen haben. Eine unzureichende Diagnostik und Probleme bei der Therapie haben dazu gefuhrt, dafi immer mehr Patienten mit gestorter zellularer Abwehr bei der Obduktion Candida albicansHerde aufweisen. Trotz guter Erfolge in der Therapie durch die neuen Triazole bleibt das Medikament der ersten Wahl Amphotericin B. Dieses sehr unvertragliche Praparat darf nur u n ter strenger Risikoabwagung verabreicht werden. Hoechst AG, Frankfurt/Main-Hochst, Germany. Correspondence: Dr Heinz Hanel, Hoechst AG, P.O. Box 800320, D-W-6230 Frankfurt/Main 80, Germany.
Es besteht demzufolge ein dringender Bedarf an einer Entwicklung vertraglicher und fungizider Arzneimittel fur tiefe Candidosen. Bisher wurden die Testsubstanzen im letalen Mausemodell gepriift. Hierbei kam es etwa 2-5 Tage nach intravenoser Infektion mit den Hefepilzen zum Tod der Tiere durch Nierenversagen. Die Versuchstiere hatten insbesondere wahrend der letzten 2 Lebenstage eine hohe Belastung durch Schmerzen und Leiden. Dies zeigte sich am gestraubten Fell und a n Apathie. Wir stellen hier ein Model1 vor, in dem ein Candida albicans-Stamm ausgewahlt wurde, der bei den Tieren weder zu erkennbaren Verhaltensauffalligkeiten, noch zu dem sonst aufgetretenen gestraubten Fell fuhrt. Trotzdem zeigen die durchgefuhrten Therapieversuche mit dem neuen Pilzstamm, daR das Ansprechen auf eine breite Palette von Handelszubereitungen genau dem klinischen Bild entspricht. Die Methode eignet sich somit, klinisch relevant verschiedene Verbindungen auf ihren systemischen Effekt gegeniiber tiefen Candidosen zu prufen, ohne die Versuchstiere dem bisherigen Leidensdruck aussetzen zu mussen.
Introduction Murine models for the evaluation of antifungal agents are generally accepted. Fatal infections with mice can readily be produced by injecting between lo5 and lo7 viable yeast cells per mouse intravenously (for an extensive review see Ref. 1). However, we have some doubts about using an inoculum which causes death in sometimes
84
H. HANELET AL.
less than 4 days. With low kidney counts [2] high inocula may lead to an obstructive mechanism with misleading consequences for the evaluation of antimycotic agents. It has been suggested that one should wait for as long as 70 days to consider a drug as active in terms of preventing lethality [3]. In accordance with Ryley [4], we suggest that lower inocula with less pathogenic strains .are helpful to evaluate therapeutic properties of the used antifungals rather then just cause slightly extended survival rates. Additionally the German “Tierschutzgesetz” (law of animal protection) asks for evaluation methods which cause as little suffering and irritation of the experimental animals as possible. We therefore present the results from studies of a large selection of Candida albicans strains which were investigated for their ability to cause persistent kidney infections without obvious signs of fatigue in the mice. Fluconazole, itraconazole, ketoconazole and amphotericin B were used in this model to evaluate the comparability with the lethal types of infections reported previously [5, 61.
obtained from Sigma Corp. (Munchen). I traconazole was a generous gift from Janssen and fluconazole was kindly supplied by Pfizer. In vivo evaluation All strains were tested using four different inocula: 1 x lo5, 3 x lo5, 6 x lo5 and 1 x lo6 CFU/animal given in 0.2 ml of physiological NaCl solution intravenously into the tail vein. Mortality and appearance of the mice was recorded daily for 21 days. T h e treatment scheme of animals for the various antimycotics is indicated in Table 5. Treatment was done twice daily beginning with D- 1 to D + 2 (D 3 respectively). Infection controls were treated with the solvent accordingly. At each day beginning one day after infection two animals from each group, treated and untreated, were killed, the kidneys dissected, weighed, homogenized and plated as described below. Kidneys from the treated group were taken, starting with D + 3 (Table 3).
+
Homogenization of kidneys and evaluation of CFU
Materials and methods Candida albicans strains Four strains of Candida albicans were selected from 150 strains obtained by a centre for laboratory medicine in Germany. The isolates were cultured from stool samples. I n previous experiments we investigated a number of parameters including mortality of mice, phospholipase production, and left and right kidney counts after intravenous infection among others [7]. One C. albicans strain (1 75) had been our internal standard for intravenous infection; it was obtained 15 years ago from Hoffmann La Roche, and was originally isolated from a patient with a systemic candidosis. All selected strains (Nos 352, 371, 410 and 420) belong to serotype A (for a discussion of the different infectivities between serotypes A and B see Ref. 8).In vitro evaluation of the phospholipase activity was done with the agar-plate method described by Price el al. [9]. The phospholipase activity of all strains was compared using strain 175 as a standard (Table 1). With this method we can evaluate phospholipase B (PLB) (and most likely the lysophospholipases) as has been published [ lo].
Compounds Amphotericin B was used in its commercial formulation Amphotericin B@and ketoconazole was
Both kidneys of each mouse were homogenized in 10 ml of physiological NaCl solution using an Ultra Turrax@ (Buhler, Germany) blender at 20,000 rpm for 1 min. The undiluted mixture was then plated on malt-agar plates containing 1% streptomycin and 1% penicillin using a Spiral Plate@ (Spiral Systems Inc., USA) which applies 92 p1 onto the plates. After incubation of the plates at 36°C for 2 days, colonies were counted according to the evaluation scheme for the Spiral Plater@.
Additional tests with strain 352 Compounds tested with strain 352 were fluconazole, ketoconazole, itraconazole (all orally) and amphotericin B (i.p.) each with 2.5 and 1.25 mg kg-’ eight times. In a second set of experiments fluconazole 6 x (2.5 mg kg-’) was compared with fluconazole 8 x (2.5 mg kg-’) using strain 352.
Results Table 1 indicates a comparison between the in vilro detected phospholipase activity and the mean survival time of mice after challenge with the five selected strains. I t is evident that among these strains there is no linear relationship between PLB activity and speed of lethality. mycoses 35, 83-88 (1992)
EXPERIMENTAL CANDIDOSIS AVOIDING
and lethality in mice after i.v. injection of C. ulbicunr suspensions (strain 175: 6 x lo5 CFU/animal) Phospholipase
B activity (Yo)
175 352 37 1 410 420
100 66 111 96 40
+
Mean survival time (days) (10 mice each) RfSD 2.2 f0.6 6.5 f2.0 5.0 f 1.8 5.0f3.1 7.0f2.0
Strain 352, having a relatively low PLB production, led to a longer survival time than strain 371 which had shown the highest PLB activity among the five isolates. Table 2 indicates the CFU counts within the homogenized kidneys of the infected mice. All animals received placebo treatment only. Strain 175 produced lower counts after the highest (1 x lo6 CFU) inoculation compared with 3 x lo5 and 6 x lo5, especially at D + 4. CFU after 1 x lo5 were minimal. Strain 352 led to small CFU counts after 1 x lo5 and 3 x lo5 but high counts were achieved after 6 x lo5 and 1 x lo6. Strain 371 behaved similarly to 352 with lower counts after 1 x lo5. Strains 410 and 420 produced only rather limited counts irrespective of the inoculum used.
Table 2. CFU of Cundida albicuns in kidneys of mice infected with five different strains from 1 to
85
Table 3 gives the data from the fluconazoletreated groups. Most fluconazole-treated mice had dramatically reduced CFU values in the kidneys due to the treatment. Strain 175 was generally reduced from D + 3 to D 4 by 99% or more. At D + 5 , the death of the infection control animals meant that the reduction could not be evaluated. Strains 352 and 371 showed a decreasing reduction a t D + 4 and D + 5 after termination of the treatment. Strain 410 caused higher CFU values than strain 420. Correspondingly, the reduction of 410 was generally between 87 and 98%) irrespective of time and infection dose. Strain 420 showed varying reductions with a peak at D + 5 . A summary of the data for illness is shown in Table 4. Strain 175 led to a distinctive mortality beginning as early as D+2. Mice infected with strain 175 showed the highest scores of illness throughout the experiment. Strains 352 and 371 led to single mortalities whereas strains 410 and 420 did not kill any mice within the 6-day scoring time. The score of illness increased with the inoculum except for strain 420 where mice appeared more ill after 3 x 1O5 than after 6 x 1O5 CFU/mouse. Taking all parameters together (CFU/mouse, score of illness, PLB production and therapeutic experiments) we selected strain 352 for further use in a mouse model, generally applying 6 x lo5 CFU by the intravenous route. Treatment tests with strain 352 are shown in
Table 1. Evaluation of phospholipase B activity in uifro
Strain no.
LETHALITY
to
5 days after infection (D + 1
D+5)
Strain no. 175
352
37 1
410
420
Inoculum/ mouse 1 x 105 3~ 105 6 x lo5 1 x 106 1 x 105 3 x 105 6 x lo5 1 x 106 1 x 105 3~ 105 6 x lo5 1 x lo6 1 x 105 3 x 105 6 x lo5 1 x 106 1 x lo5 3~ 1 0 5 6 x lo5 1 x lo6
mycoses 35, 83-88 (1992)
Observed CFU x lo5/" kidneys
D+ 1
D+2
D+3
D+4
D+5
0.03 0.4 0.1 1 4.1 1 0.06 0.27 0.76 1.11 0.12 0.33 0.23 0.35 0.008 0.052 0.036 0.48 0.012 0.05 0.027 0.19
0.36 0.93 3.76 6.44 0.39 1.47 36.6 70.0 0.23 0.49 70.0 70.0 0.081 0.49 0.78 1.5 0.095 0.03 0.046 1.9
0.074 5.1 12.0 12.2 0.7 1.3 70.0 38.5 0.06 35.3 70.0 0.6 1 0.42 2.91 0.6 I .46 0.06 0.002 0.002 4.07
0.64 39.0 38.0 0.15 0.90 1.62 4.78 43.8 0.36
0.22 1.85 dead dead 0.26 7.7 10.4 39.2 0.23 0.14 0.59 35.6 3.6 8.5 25.2 I .48 0.03 0.02 0.012 35.8
0.27
35.5 35.3 1.44 2.37 1.94 1.3 0.19 0.022 0.012 0.56
86
H.
H A N E L ET AL.
Table 3. CFU of Cundidu nlbicuns in kidneys of mice treated orally eight times with fluconazole 2.5 mg kg-’ after infection with five different strains from four different inocula Strain no.
175
352
37 1
410
420
D+3
Inoculum/ mouse
Ix 3~ 6x 1x Ix 3~
105 105 106
105 105 6 x lo5 1 x 106 I x 105 3~ 105 6 x lo5 1 x 106 I x 105
3~ 6x 1x Ix 3~ 6x 1x
CFU
(YO)
CFU
0.0
100 99.76 99.79 99.48 98.71 99.04 99.98 99.96 89.66 99.96 99.96 91.19 89.66 98.69 93.65 96.38 95.0 50.0 50.0 99.88
0.04 0.19 0.18 2.97 0.11 1.61 2.65 1.31 0.09 0.18 0.57 0.72 0.38 1.24 1.81 0.83 0.0 0.04
0.12 0.25 0.64 0.09 0.12 0.15 0.16 0.06 0.15 0.25 0.54 0.17 0.38 0.38 0.53 0.03 0.01 0.01 0.05
lo5
105 lo5 lo6 105 105 lo5 lo6
D+4
0.0 1 0.02
I
D+5 (YO)
CFU
(%I
99.37 99.95 99.95 0.0 98.78 90.06 94.46 99.70 97.52 93.38 99.84 99.80 97.36 94.76 90.66 93.59 100 81.82 91.67 99.64
0.28 2.1 1 2.15 1.32 1.26 1.77 1.42 4.07 0.17 0.15 0.32 0.76 1.48 1.18 5.08 1.89 0.0 0.01 0.01 0. I
87.27 88.56 r.d. r.d. 51.54 97.70 98.63 98.96 92.70 89.05 94.58 99.79 95.89 98.61 97.98 87.23 I00 99.47 91.67 99.97 I
CFU= CFU x 104/2kidneys;
(Yo)= Yo inhibition;
r.d. =respective control dead.
Table 4. Score values of ‘illness’ in mice infected intravenously with five different Cundidu ufbicuns strains one to five days after infection Strain no.
Inoculum
D+1
D+2
D+3 D+4
D+5
Table 5. It is obvious that all of the antimycotics used are effective to a varying degree, with amphotericin B (ip.) being the most effective,
followed by fluconazole, itraconazole and ketoconazole (orally).
Discussion Intravenous inoculation of mice with Candida albicans leads to graded fungal kidney counts. As Ryley et al. [3] demonstrated, high inocula (3 x lo6) lead to early death with low counts, whereas moderate inocula (3 x lo5) produced high counts in infections which some mice survived. The comparison of various C. albicans strains in our infection model reflects the need for a predictable method which avoids the early death due to kidney failure. Strain-dependent differences in susceptibility of mice were investigated by Marquis et al. [ 111. The authors mainly focused on the kidney weight as a parameter of infection. We used the fungal count only, for we could not see a definite correlation between the weight of a n infected kidney and the number of colony-forming units ([7] and unpublished results). This is in accordance with the histopathological investigations by Blyth [ 121 and Ryley & Ryley [13], both of whom demonstrated the different cellular reaction of kidneys towards blastoconidia and filaments which then lead to different kidney weights. Comparisons of different strains (wild types and mutants) have been made mycoses 35, 83-88 (1992)
EXPERIMENTAL CANDIDOSIS
87
AVOIDING LETHALITY
Table 5. Influence of treatment regimen on the number of CFU/kidney at D + 3 after Candida albicans intravenous infection of mice with strain 352 (6 x lo5 CFU/mouse)
Compound
Applications
Dose
(mg kg-’) Fluconazole (oral) Ketoconazole (oral) I traconazole (oral) Amphotericin B (i.P.) Infection control
1.25 2.5 2.5 1.25 2.5 1.25 2.5 1.25 2.5
8 6 8 8 8
a
8 8 8 8
but not in view of a decreased suffering [14]. The five strains investigated in our experiments differed in their ability to produce high kidney counts. As can be seen from the phospholipase comparison (Table 1) this cannot be the only factor determining pathogenicity. Besides phospholipase a number of other pathomechanisms have been discussed (for a recent review see Refs 15 and 16). As early as 1940, Henrici [I71 postulated an exotoxin produced by Candida albicans to explain effects he observed in experimental infections. Irrespective of the unresolved problems concerning the pathomechanism, we focused on the behaviour of the infected mice in order to prevent suffering of the animals. Table 4 indicates that our previous reference strain 175 led to early symptoms which were reflected by a wet appearance of the fur and loss of appetite. Strain 410 showed the least severe symptoms but failed to produce constant and reliable kidney counts. It is interesting to note that the CFU reduction due to fluconazole at D 5 was stronger than for the more aggressive strain 175, probably due to a slower tissue invasion and a different morphological appearance. It is generally accepted that the fungistatic azole compounds inhibit mycelia at a lower concentration than they do blastoconidia. We therefore selected strain 352 with a relatively low phospholipase activity for further evaluation in therapeutic experiments. We did not try to modulate the response of the host with antibiotics [18] or silica to modify the macrophage response [ 191 because a routine model for the evaluation of antifungal agents has to reflect the clinical situation as closely as possible. This is in accordance with the experiments of Hare & Loebenberg [20] who also used kidney counts for distinguishing between fungicidal and fungistatic compounds. Our results with fluconazole with the five strains show that the recent
+
mycoses 35, 83-88 (1992)
Mean CFU/kidney
Reduction
Mice
( x 103)
(Yo)
(n)
8.10 6.93 2.26 1512.12 330.82 115.16 26.23 1.61 0.48 1663.95
99.5 99.6 99.9 9.0 80.1 93.1 98.4 99.9 99.97
5 10
0.0
10
5 5 5 5 5 5 10
triazoles can reduce the colony-forming units dramatically under the experimental conditions tested. However, as Hare & Loebenberg [20] indicated, we observed high mortality after the termination of treatment with triazoles. Amphotericin B, on the other hand, obviously acts by the fungicidal way under these experimental conditions. We therefore suggest the screening of potential antifungal compounds with the short model first, evaluating the fungistatic properties. Suitable drugs should then be tested again by observing the treated mice for at least two months to investigate the fungicidal properties of the selected compounds. The development of an infection model in which the mice suffer to only a limited degree is necessary for the ethical justification for undertaking animal experiments in the routine search for new antifungal agents.
References 1 Miyaji, M. (1987) Animal Models in Medical Mycology. Boca Raton, Florida: C R C Press. 2 Ryley, J. F., McGregor, S., Lister, S. C. & Jackson, K. P. (1988) Kidney function in experimental systemic candidosis of mice. Mycoses 31, 203-207. 3 Ryley, J. F., McGregor, S. & Wilson, R. G. (1988) The activity of ICI 195739, a novel orally active bis-triazole, in rodent models of fungal and protozoal infections. Ann. N.Y. Acad. Sci. 544, 310-328. 4 Ryley, J. F. (1990) Screening and evaluation in vivo. Handbook of Experimental Pharmacology 96, 1 29- 147. 5 Troke, P. F., Andrews, R. J., Brammer, K. W., Marriott, M. S. & Richardson, K. (1985) Efficacy of UK-49,858 (Fluconazole) against Candida albicans experimental infections in mice. Antimicrob. Agents Chemother. 28, 8 I 5-8 18. 6 Ryley, J. F., & McGregor, S. (1988) A multi infection model for antifungal screening in vivo. 3. Antimicrob. Chemother. 22, 353-358. 7 Hanel, H., Schiitz, K. D., Buslau, M. & Holzmann, H. (1 990) Untersuchungen von Candida albicans-Isolaten aus Stuhlproben von Neurodermitikern. 24th Scientific Confer-
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11
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H. HANELET AL.
me of the German-Speaking Mycological Society, Gottingen, Abstract No 24, p. 9. Polak, A., Odds, F. C., Liidin, E. & Scholer, H. J. (1985) Correlation of susceptibility test results in vitro with response in vivo: Ketoconazole therapy in a systemic candidiasis model. Chemotherapy 31, 395-404. Price, M. F., Wilkinson, I. D. & Gentry, L. 0. (1982) Plate method for detection of phospholipase activity in Candida albicans. Sabouraudia 20, 7- 14. Hanel, H., Menzel, I., Buslau, M. & Holzmann, H. (1989) Messung der Phospholipaseaktivitat von Cundidu albicans im Darm. In: Miiller, J., Ottenjann, R. & Seifert, J. (eds) Okosystem Dorm. Berlin: Springer, pp. 185-187. Marquis, G., Montplaisir, S., Pelletier, M., Mousseau, S. & Auger, P. (1986) Strain-dependent differences in susceptibility ofmice to experimental candidosis. 3.Infect. Dis. 151, 906-909. Blyth, W. (1959) Hostlparasite relationships in experimental moniliasis. Mycopath. Mycologia Applicata 10, 269-282. Ryley, J. F. & Ryley, N. G. (1990) Cundida albicansdo mycelia matter? Sabouraudia 28, 225-239.
14 Fromtling, R. A., Abruzzo, G. K., Edison, A. & Manning-Zweerink, W. (1988) Renal pathology and spleen cell chemiluminescence of mice infected with a wild-type and a low-virulence mutant of Candidu albicans. Zbl. Bakt. Hyg. A 268,405-415. 15 Ghannoum, M. A. & Abu-Elteen, K. H. (1990) Pathogenicity determinants of Candida. Mycoses 33, 265-282. 16 Odds, F. C. (1988) Candida and Candidosis. 2nd edn. London: Baillitre Tindall. 17 Henrici, A. T. (1940) 3. Bact. 39, 113-citated after Winner, H. I. (1958) An experimental approach to the study of infections by yeast-like organisms. Proc. Royal SOC.Med. 51, 496-499. 18 Blyth, W. (1958) The influence of antibiotics on experimental moniliasis. Mycopath. Mycologia Applicata 10, 91-1 12. 19 Sinha, B. K., Monga, D. P. & Prasad, S. (1987) Studies on the role of macrophages in experimental candidosis in mice. Mycoscs 30, 105-1 12. 20 Hare, R. S. & Loebenberg, D. (1988) Animal models in the search for antifungal agents. Am. SOC.Microbiol. NCWS 54, 235-239.
mycoses 35, 83-88 (1992)
