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Efficacy of intrathecal administration of liposomal amphotericin B combined with voriconazole in a murine model of cryptococcal meningitis
International Journal of Antimicrobial Agents 39 (2012) 223–227
Contents lists available at SciVerse ScienceDirect
International Journal of Antimicrobial Agents journal homepage: http://www.elsevier.com/locate/ijantimicag
Efficacy of intrathecal administration of liposomal amphotericin B combined with voriconazole in a murine model of cryptococcal meningitis Alexandra F. Gazzoni a , Javier Capilla b , Emilio Mayayo a , Josep Guarro b,∗ a b
Pathologic Anatomy Unit, Facultat de Medicina i Ciències de la Salut, IISPV, Universitat Rovira i Virgili, Reus, Spain Microbiology Unit, Facultat de Medicina i Ciències de la Salut, IISPV, Universitat Rovira i Virgili, Reus, Spain
a r t i c l e
i n f o
Article history: Received 9 May 2011 Accepted 21 October 2011 Keywords: Cryptococcosis Cryptococcus neoformans Meningitis Mouse Liposomal amphotericin B Intrathecal
a b s t r a c t Meningitis is one of the most fatal manifestations of cryptococcosis, even with specific treatment. Combination of a prompt diagnosis and appropriate therapy are critical to reduce the fungal load and the inflammatory response effects of the proliferation of yeast into the central nervous system (CNS). Mice with experimental acute meningitis caused by Cryptococcus neoformans were treated with liposomal amphotericin B (L-AmB) administered intrathecally (i.t.c.) at 0.006 mg/kg weekly or intravenously (i.v.) at 10 mg/kg daily or with voriconazole (VCZ) administered orally at 30 mg/kg per dose twice daily or with combinations of both drugs, i.e. L-AmB i.t.c. + VCZ or L-AmB i.v. + VCZ at the same doses as used in the monotherapies. All treatments significantly increased the survival of animals in comparison with the control group, with VCZ being less effective in comparison with all other treatments (P ≤ 0.012). All treatments, with the exception of VCZ (P = 0.533), reduced fungal burdens in the brain in comparison with controls. The combination of L-AmB i.t.c. + VCZ showed a synergistic effect in the reduction of fungal load that was significantly superior to any tested therapy (P ≤ 0.039). Histologically, untreated animals showed a marked inflammatory response with massive fungal cells in the meninges, whilst treated animals showed a variable number of fungal cells in the CNS, with the exception of animals receiving L-AmB i.t.c. + VCZ in which neither yeasts nor inflammation were observed. © 2011 Elsevier B.V. and the International Society of Chemotherapy. All rights reserved.
1. Introduction Cryptococcus neoformans is a common pathogenic fungus with a worldwide distribution and is able to cause severe and often fatal infections in humans. Although clinical manifestations of human cryptococcosis are variable, the skin, lungs and brain are amongst the major sites of infection. Neurological manifestations, including meningitis and, less commonly, parenchymal central nervous system (CNS) granulomatous disease, are the most severe complications of cryptococcosis [1]. Without specific treatment, cryptococcal infection involving the CNS shows mortality rates near to 100%, generally within 2 weeks after clinical presentation [2]. Advances in earlier diagnosis and antifungal treatment have improved the clinical outcome of such disease in recent years; however, the mortality rate, especially that associated with CNS cryptococcosis, is still high [3] and in 20–40% of patients
∗ Corresponding author. Present address: Unitat de Microbiologia, Facultat de Medicina, Universitat Rovira i Virgili, Carrer Sant Llorenc¸ 21, 43201 Reus, Spain. Tel.: +34 977 759 359; fax: +34 977 759 322. E-mail address: [email protected] (J. Guarro).
suffering cryptococcal meningitis treatment fails even after long periods [4,5]. The gold-standard treatment of CNS-related cryptococcosis includes induction with amphotericin B deoxycholate (AmBD) with or without flucytosine (5FC), followed by a maintenance course with fluconazole (FLZ) or itraconazole [3]. Unfortunately, antifungal therapy is not always successful (40–60% favourable response) and in addition AmBD may be limited by its acute nephrotoxicity, which can be reduced by using lipid formulations of amphotericin B such as AmB colloidal dispersion or liposomal AmB (L-AmB) [3,5]; however, these formulations are generally unable to pass through the blood–brain barrier. In CNS fungal infections, the inflammatory process enhances the permeability to L-AmB [4], allowing higher drug levels in nervous tissues and consequently increasing the efficacy. However, there is a higher risk of death due to the elevation in intracranial pressure associated with inflammation [6]. FLZ treatment must be carefully monitored due to isolation of resistant strains, which limits its use and clinical efficacy [7]. For these reasons, it is necessary to explore new strategies aimed at achieving an effective treatment against this disease. Recent studies have demonstrated the in vitro activity of the triazoles posaconazole, voriconazole (VCZ) and isavuconazole against C. neoformans, even against isolates with reduced susceptibility to FLZ [8–10].
0924-8579/$ – see front matter © 2011 Elsevier B.V. and the International Society of Chemotherapy. All rights reserved. doi:10.1016/j.ijantimicag.2011.10.014
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Fig. 1. Survival of OF-1 mice infected intracranially with 580 colony-forming units (CFU) of Cryptococcus neoformans. Treatments consisted of liposomal amphotericin B (LAmB) administered intrathecally (i.t.c.) at 0.006 mg/kg once weekly for 3 weeks, L-AmB administered intravenously (i.v.) at 10 mg/kg/day or voriconazole (VCZ) administered orally twice daily by gavage at 60 mg/kg/day. Combination therapies consisted of L-AmB i.t.c. + VCZ or L-AmB i.v. plus VCZ administered as in monotherapies. All treatments started 5 days after infection and continued until Day 19 post infection.
However, scarce data exist about their efficacy in animal models or in clinical practice. Administering drugs directly into the CNS could be an alternative, as occurs in other fungal infections that severely affect the CNS such as coccidioidomycosis, and especially in those cases where conventional therapy fails or a relapse of the infection occurs. In this study, the in vivo efficacy of intrathecal administration of L-AmB alone and in combination with VCZ was compared using a previously described murine model of cryptococcal meningitis caused by C. neoformans [11].
2. Materials and methods 2.1. Strain A clinical isolate of C. neoformans var. neoformans (serotype AD), strain FMR 8398, obtained from the cerebrospinal fluid of a human immunodeficiency virus (HIV)-positive patient, was used in the present study. The previously determined minimum inhibitory concentrations of AmB and VCZ for this strain were 0.5 g/mL and 0.03 g/mL, respectively [11]. The fungus was grown on Sabouraud dextrose agar (SDA) (Laboratorios Conda, S.A., Madrid, Spain) and was incubated at 35 ◦ C for 48 h.
2.2. Inoculum On the day of infection, a single colony was re-suspended in sterile saline and was adjusted to 11.5 × 103 yeasts/mL by dilution and haemocytometer counting. Viability was confirmed by placing 10-fold dilutions onto SDA plates and incubation for 5 days at 35 ◦ C, with the resulting number of colony-forming units (CFU) being 11.6 × 103 CFU/mL.
2.3. Infection model Four-week-old OF-1 male mice were anaesthetised by inhalation of fluothane vapours (0.5%). A total of 580 CFU/mouse [11] in a volume of 50 L was inoculated intracranially at the midline point on the cranium, 4–5 mm posterior to the eyes [12,13]. Mice were fully recovered from the procedure without neurological sequelae. Animal research procedures were approved by the Animal Welfare Committee of the Rovira i Virgili University (Reus, Spain).
2.4. Antifungal therapy Groups of 10 mice (5 per cage) were randomly assigned. All treatments began 5 days after infection and were continued for 15 days. Animals received the following monotherapies or combined therapies: L-AmB (AmBisome; Gilead Sciences Inc., Dublin, Ireland) at 10 mg/kg given intravenously (i.v.) into the lateral tail vein once daily; L-AmB at 0.006 mg/kg given intrathecally (i.t.c.) once a week for 3 weeks; VCZ (VFEND® ; Pfizer Inc., Madrid, Spain) at 30 mg/kg administered orally (p.o.) by gavage twice daily; L-AmB i.t.c. + VCZ p.o.; or L-AmB i.v. + VCZ p.o. The doses and schedule used in the combined therapies were the same as in the monotherapies. Control animals received a placebo of 50 L consisting of 0.9% saline administered i.t.c. once weekly for 3 weeks. From 1 day prior to the infection, mice receiving VCZ were given grapefruit juice (50% in water) instead of water [14]. Intrathecal administration of L-AmB was performed under anaesthesia induced by xylazine (10 mg/kg) and ketamine (100 mg/kg), both given intraperitoneally. A superficial skin incision was made over the lumbar vertebrae and the drug was delivered in a volume of 50 L into the subarachnoid space via lumbar puncture into the interspaces between the first and second lumbar vertebrae using a U-100 insulin syringe with a 27 G needle (Terumo Europe, Leuven, Belgium). After dosage, the skin incision was closed with wound clips. Mice were observed until they recovered from anaesthesia, at which time they showed no evidence of neurological abnormalities. Animals were checked twice daily and those showing signs of discomfort or survivors 21 days post infection were euthanised by CO2 anoxia. 2.5. Tissue burden At 48 h after the end of the therapy (i.e. Day 21 post infection), treated animals that survived were euthanised as described and their brains were removed. Owing to the quick death in untreated animals, euthanised animals from the control group were included on Days 11 and 12 post infection in the tissue burden study. Onehalf of the organ was weighed and mechanically homogenised in 0.9% saline. The homogenates were 10-fold diluted, placed onto SDA plates and incubated at 35 ◦ C for 72 h for CFU determination. 2.6. Histopathological studies The remaining half of the brain was placed in 10% buffered formalin. Samples were dehydrated, paraffin embedded, sliced into
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2.7. Statistical analysis Comparisons of survival were done by log-rank test and comparisons of fungal burden by Mann–Whitney U-test applying Bonferroni correction for multiple comparisons. All comparisons were done using GraphPad 4.0 for Windows (GraphPad Software Inc., La Jolla, CA). 3. Results 3.1. Survival
Fig. 2. Scattergram and median (horizontal bar) of colony-forming units (CFU) recovered from brains 21 days after intracranial infection with 580 CFU of Cryptococcus neoformans. Treatments consisted of liposomal amphotericin B (L-AmB) administered intrathecally (i.t.c.) at 0.006 mg/kg once weekly for 3 weeks, L-AmB administered intravenously (i.v.) at 10 mg/kg/day or voriconazole (VCZ) administered orally twice daily by gavage at 60 mg/kg/day. Combination therapies consisted of L-AmB i.t.c. + VCZ or L-AmB i.v. plus VCZ administered as in monotherapies. All treatments started 5 days after infection and continued until Day 19 post infection. Data from control group correspond to animals euthanised 11 days and 12 days post infection.
2 m sections and stained with haematoxylin and eosin, periodic acid-Schiff and Grocott’s methenamine silver stain. A pathologist blind to the treatment each animal had received carried out the microscopic examination of sections.
All control animals succumbed to the infection within 9–12 days post infection, but animals receiving monotherapy (VCZ, LAmB i.v. or L-AmB i.t.c.) or combination therapy (L-AmB i.v. + VCZ or L-AmB i.t.c. + VCZ) survived significantly longer (P ≤ 0.05) than control mice (Fig. 1). No differences were observed amongst therapies, with the exception of VCZ administered alone, which was significantly less effective in prolonging survival than the other treatments (P ≤ 0.012). 3.2. Fungal load The tissue burden study (Fig. 2) showed that control animals had a high fungal load in the brain (median log10 ± standard deviation 6.883 ± 0.306). VCZ administered alone failed to reduce tissue burden in comparison with the control group (P = 0.533), whilst LAmB administered i.t.c. or i.v. significantly reduced burdens with respect to the controls (P = 0.03 and 0.02, respectively). Combination of L-AmB i.t.c. or i.v. with VCZ showed a reduction in CFU counts compared with the untreated animals (P = 0.014 and 0.04, respectively), but the combination of L-AmB i.v. + VCZ did not show an advantage in reducing burdens over the monotherapy with
Fig. 3. Brain sections of mice infected intracranially with 580 colony-forming units (CFU)/animal. (A) Control animals showing enlargement of the meninges with massive inflammatory cells and encapsulated yeasts (arrows). (B) Animals treated with oral voriconazole (VCZ) showing enlargement of the meninges with abundant inflammatory cells, fungal elements and vascular congestion. (C) Animals treated with liposomal amphotericin B (L-AmB) intrathecally (i.t.c.) showing discrete enlargement of the meningeal structure and scarce yeasts. (D) Animals treated with L-AmB i.t.c. plus VCZ showing absence of fungal cells, discrete enlargement of the meninges and vascular congestion with oedema. Haematoxylin and eosin, ×250.
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L-AmB i.v. (P = 0.505). However, combination of L-AmB i.t.c. with VCZ resulted in a great reduction of fungal load in comparison with L-AmB i.t.c. alone (P = 0.039). This combination showed the greatest reduction in CFU counts, being significant in comparison with all the other therapies (P ≤ 0.039). 3.3. Histology findings The histopathological study showed extensive damage in the meningeal membranes of control animals. Macroscopically, they showed a mucous white-like layer covering the brain. Microscopically, control animals showed enlargement of the meninges with oedema and abundant inflammatory and meningeal reactive cells. In addition, numerous globose encapsulated fungal cells invading the meninges were also present (Fig. 3A). Despite the extensively damaged meninges, no cerebritis was observed and only scarce or no fungal cells were noticed in the brain parenchyma. Monotherapy-treated animals showed different degrees of meningitis with the presence of few fungal cells (Fig. 3B and C). In animals receiving L-AmB i.t.c. + VCZ, no fungal cells were observed and the meninges were apparently normal with the exception of small foci with evidence of tissue recovery with discrete oedema as a result of slight vascular congestion (Fig. 3D). 4. Discussion Meningitis is one of the most fatal complications of cryptococcosis in patients with acquired immune deficiency syndrome (AIDS) [15–17], although it has been also sporadically reported in non-HIV patients. These patients are frequently affected by the adverse side effects caused by administering antifungal treatments, especially with AmB and 5FC [18–20]. In cases of acute meningitis, trying to reduce the high burden in nervous tissue is crucial for improving the clinical outcome. Despite standard therapy, a growing number of reports have indicated that standard therapy is ineffective in some patients [3,21]. This has led to the need for more effective therapies for cryptococcosis affecting the CNS. VCZ has been suggested as an alternative therapy, and animal models of cryptococcal meningitis have demonstrated a reduction in fungal load and prolongation of survival after VCZ administration [22,23]. Although clinical and experimental data have shown VCZ as an alternative against cryptococcal meningitis, serum levels of this drug are highly variable amongst adults [24]. In the present study, the efficacy of L-AmB in achieving a fast reduction of CFU in CNS tissues was tested using an advanced acute model of meningitis in mice. Previous experimental studies conducted on rabbits have shown that continual intrathecal administration of lipidic AmB at concentrations ranging from 0.0015 mg/kg to 0.045 mg/kg did not show any signs or symptoms of neurological or toxic effects in the animals [25]. Although intrathecal administration is not the usual route of administration for AmB or its lipidic derivatives, this route is used in the treatment of coccidioidal meningitis in humans. Because coccidioidal meningitis cannot be cured with available antifungal agents, guidelines recommend therapy with AmB i.t.c. when consolidation therapy with azoles fails, at doses ranging from 0.01 mg to 1.5 mg and at intervals ranging from daily to weekly [26,27]. Few clinical studies have been conducted on the efficacy of intrathecal administration of antifungal agents against cryptococcal meningitis. However, in one of them, 22 non-HIV patients with meningitis caused by Cryptococcus were cured with AMB i.t.c. without any recurrence [28]. The current results demonstrate that L-AmB i.t.c. combined with VCZ significantly reduced the number of yeasts in the brain in comparison with the other therapies tested. In addition, histopathological findings showed that animals receiving such treatment had normal
meninges. The good distribution into the CNS of VCZ, which is not dependent on meningeal inflammation, is an additional advantage [10,29], making the combination of both drugs a potential alternative in salvage treatment of cryptococcal meningitis. Since CNS involvement has been documented in 52–61% of transplant recipients with cryptococcosis, and 25% of them have renal dysfunction at the time of diagnosis [17], the use of L-AmB i.t.c. at low doses could be considered a therapeutic approach in those patients, especially when systemic antifungal agents fail. Funding: This work was partially funded by the Universitat Rovira i Virgili (Reus, Spain). Competing interests: None declared. Ethical approval: Animal research procedures were approved by the Animal Welfare Committee of the Rovira i Virgili University (Reus, Spain).
References [1] Eric Searls D, Sico JJ, Bulent Omay S, Bannykh S, Kuohung V, Baehring J. Unusual presentations of nervous system infection by Cryptococcus neoformans. Clin Neurol Neurosurg 2009;111:638–42. [2] French N, Gray K, Watera C, Nakiyingi J, Lugada E, Moore M, et al. Cryptococcal infection in a cohort of HIV-1-infected Ugandan adults. AIDS 2002;16: 1031–8. [3] Perfect JR, Dismukes WE, Dromer F, Goldman DL, Graybill JR, Hamill RJ, et al. Clinical practice guidelines for the management of cryptococcal disease: 2010 update by the Infectious Diseases Society of America. Clin Infect Dis 2010;50:291–322. [4] Takemoto K, Yamamoto Y, Ueda Y. Influence of the progression of cryptococcal meningitis on brain penetration and efficacy of AmBisome in a murine model. Chemotherapy 2006;52:271–8. [5] Walsh TJ, Finberg RW, Arndt C, Hiemenz J, Schwartz C, Bodensteiner D, et al. Liposomal amphotericin B for empirical therapy in patients with persistent fever and neutropenia. National Institute of Allergy and Infectious Diseases Mycoses Study Group. N Engl J Med 1999;340:764–71. [6] York J, Bodi I, Reeves I, Riordan-Eva P, Easterbrook PJ. Raised intracranial pressure complicating cryptococcal meningitis: immune reconstitution inflammatory syndrome or recurrent cryptococcal disease? J Infect 2005;51: 165–71. [7] Armengou A, Porcar C, Mascaro J, Garcia-Bragado F. Possible development of resistance to fluconazole during suppressive therapy for AIDS-associated cryptococcal meningitis. Clin Infect Dis 1996;23:1337–8. [8] Thompson 3rd GR, Wiederhold NP, Fothergill AW, Vallor AC, Wickes BL, Patterson TF. Antifungal susceptibilities among different serotypes of Cryptococcus gattii and Cryptococcus neoformans. Antimicrob Agents Chemother 2009;53:309–11. [9] Messer SA, Moet GJ, Kirby JT, Jones RN. Activity of contemporary antifungal agents, including the novel echinocandin anidulafungin, tested against Candida spp., Cryptococcus spp., and Aspergillus spp.: report from the SENTRY Antimicrobial Surveillance Program (2006 to 2007). J Clin Microbiol 2009;47:1942–6. [10] van Duin D, Cleare W, Zaragoza O, Casadevall A, Nosanchuk JD. Effects of voriconazole on Cryptococcus neoformans. Antimicrob Agents Chemother 2004;48:2014–20. [11] Serena C, Pastor FJ, Marine M, Rodriguez MM, Guarro J. Efficacy of voriconazole in a murine model of cryptococcal central nervous system infection. J Antimicrob Chemother 2007;60:162–5. [12] Chiller TM, Luque JC, Sobel RA, Farrokhshad K, Clemons KV, Stevens DA. Development of a murine model of cerebral aspergillosis. J Infect Dis 2002;186:574–7. [13] Capilla J, Mayayo E, Serena C, Pastor FJ, Guarro J. A novel murine model of cerebral scedosporiosis: lack of efficacy of amphotericin B. J Antimicrob Chemother 2004;54:1092–5. [14] Graybill JR, Najvar LK, Gonzalez GM, Hernandez S, Bocanegra R. Improving the mouse model for studying the efficacy of voriconazole. J Antimicrob Chemother 2003;51:1373–6. [15] Swe Han KS, Bekker A, Greeff S, Perkins DR. Cryptococcus meningitis and skin lesions in an HIV negative child. J Clin Pathol 2008;61:1138–9. [16] Aydemir H, Pis¸kin N, Oztoprak N, Celebi G, Tekin IO, Akduman D. Cryptococcus neoformans meningitis in a HIV negative miliary tuberculosis-suspected patient. Mikrobiyol Bul 2008;42:519–24 [in Turkish]. [17] Husain S, Wagener MM, Singh N. Cryptococcus neoformans infection in organ transplant recipients: variables influencing clinical characteristics and outcome. Emerg Infect Dis 2001;7:375–81. [18] Bicanic T, Wood R, Meintjes G, Rebe K, Brouwer A, Loyse A, et al. High-dose amphotericin B with flucytosine for the treatment of cryptococcal meningitis in HIV-infected patients: a randomized trial. Clin Infect Dis 2008;47:123–30. [19] Pappas PG. Cryptococcosis in the developing world: an elephant in the parlor. Clin Infect Dis 2010;50:345–6. [20] Pasqualotto AC. Amphotericin B: the higher the dose, the higher the toxicity. Clin Infect Dis 2008;47:1110 [author reply 1111].
A.F. Gazzoni et al. / International Journal of Antimicrobial Agents 39 (2012) 223–227 [21] Saag MS, Graybill RJ, Larsen RA, Pappas PG, Perfect JR, Powderly WG, et al. Practice guidelines for the management of cryptococcal disease. Infectious Diseases Society of America. Clin Infect Dis 2000;30:710–8. [22] Kirkpatrick WR, Najvar LK, Bocanegra R, Patterson TF, Graybill JR. New guinea pig model of cryptococcal meningitis. Antimicrob Agents Chemother 2007;51:3011–3. [23] Mavrogiorgos N, Zaragoza O, Casadevall A, Nosanchuk JD. Efficacy of voriconazole in experimental Cryptococcus neoformans infection. Mycopathologia 2006;162:111–4. [24] Trifilio S, Pennick G, Pi J, Zook J, Golf M, Kaniecki K, et al. Monitoring plasma voriconazole levels may be necessary to avoid subtherapeutic levels in hematopoietic stem cell transplant recipients. Cancer 2007;109:1532–5. [25] Clemons KV, Sobel RA, Williams PL, Stevens DA. Comparative toxicities and pharmacokinetics of intrathecal lipid (amphotericin B colloidal dispersion) and
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Contents lists available at SciVerse ScienceDirect
International Journal of Antimicrobial Agents journal homepage: http://www.elsevier.com/locate/ijantimicag
Efficacy of intrathecal administration of liposomal amphotericin B combined with voriconazole in a murine model of cryptococcal meningitis Alexandra F. Gazzoni a , Javier Capilla b , Emilio Mayayo a , Josep Guarro b,∗ a b
Pathologic Anatomy Unit, Facultat de Medicina i Ciències de la Salut, IISPV, Universitat Rovira i Virgili, Reus, Spain Microbiology Unit, Facultat de Medicina i Ciències de la Salut, IISPV, Universitat Rovira i Virgili, Reus, Spain
a r t i c l e
i n f o
Article history: Received 9 May 2011 Accepted 21 October 2011 Keywords: Cryptococcosis Cryptococcus neoformans Meningitis Mouse Liposomal amphotericin B Intrathecal
a b s t r a c t Meningitis is one of the most fatal manifestations of cryptococcosis, even with specific treatment. Combination of a prompt diagnosis and appropriate therapy are critical to reduce the fungal load and the inflammatory response effects of the proliferation of yeast into the central nervous system (CNS). Mice with experimental acute meningitis caused by Cryptococcus neoformans were treated with liposomal amphotericin B (L-AmB) administered intrathecally (i.t.c.) at 0.006 mg/kg weekly or intravenously (i.v.) at 10 mg/kg daily or with voriconazole (VCZ) administered orally at 30 mg/kg per dose twice daily or with combinations of both drugs, i.e. L-AmB i.t.c. + VCZ or L-AmB i.v. + VCZ at the same doses as used in the monotherapies. All treatments significantly increased the survival of animals in comparison with the control group, with VCZ being less effective in comparison with all other treatments (P ≤ 0.012). All treatments, with the exception of VCZ (P = 0.533), reduced fungal burdens in the brain in comparison with controls. The combination of L-AmB i.t.c. + VCZ showed a synergistic effect in the reduction of fungal load that was significantly superior to any tested therapy (P ≤ 0.039). Histologically, untreated animals showed a marked inflammatory response with massive fungal cells in the meninges, whilst treated animals showed a variable number of fungal cells in the CNS, with the exception of animals receiving L-AmB i.t.c. + VCZ in which neither yeasts nor inflammation were observed. © 2011 Elsevier B.V. and the International Society of Chemotherapy. All rights reserved.
1. Introduction Cryptococcus neoformans is a common pathogenic fungus with a worldwide distribution and is able to cause severe and often fatal infections in humans. Although clinical manifestations of human cryptococcosis are variable, the skin, lungs and brain are amongst the major sites of infection. Neurological manifestations, including meningitis and, less commonly, parenchymal central nervous system (CNS) granulomatous disease, are the most severe complications of cryptococcosis [1]. Without specific treatment, cryptococcal infection involving the CNS shows mortality rates near to 100%, generally within 2 weeks after clinical presentation [2]. Advances in earlier diagnosis and antifungal treatment have improved the clinical outcome of such disease in recent years; however, the mortality rate, especially that associated with CNS cryptococcosis, is still high [3] and in 20–40% of patients
∗ Corresponding author. Present address: Unitat de Microbiologia, Facultat de Medicina, Universitat Rovira i Virgili, Carrer Sant Llorenc¸ 21, 43201 Reus, Spain. Tel.: +34 977 759 359; fax: +34 977 759 322. E-mail address: [email protected] (J. Guarro).
suffering cryptococcal meningitis treatment fails even after long periods [4,5]. The gold-standard treatment of CNS-related cryptococcosis includes induction with amphotericin B deoxycholate (AmBD) with or without flucytosine (5FC), followed by a maintenance course with fluconazole (FLZ) or itraconazole [3]. Unfortunately, antifungal therapy is not always successful (40–60% favourable response) and in addition AmBD may be limited by its acute nephrotoxicity, which can be reduced by using lipid formulations of amphotericin B such as AmB colloidal dispersion or liposomal AmB (L-AmB) [3,5]; however, these formulations are generally unable to pass through the blood–brain barrier. In CNS fungal infections, the inflammatory process enhances the permeability to L-AmB [4], allowing higher drug levels in nervous tissues and consequently increasing the efficacy. However, there is a higher risk of death due to the elevation in intracranial pressure associated with inflammation [6]. FLZ treatment must be carefully monitored due to isolation of resistant strains, which limits its use and clinical efficacy [7]. For these reasons, it is necessary to explore new strategies aimed at achieving an effective treatment against this disease. Recent studies have demonstrated the in vitro activity of the triazoles posaconazole, voriconazole (VCZ) and isavuconazole against C. neoformans, even against isolates with reduced susceptibility to FLZ [8–10].
0924-8579/$ – see front matter © 2011 Elsevier B.V. and the International Society of Chemotherapy. All rights reserved. doi:10.1016/j.ijantimicag.2011.10.014
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Fig. 1. Survival of OF-1 mice infected intracranially with 580 colony-forming units (CFU) of Cryptococcus neoformans. Treatments consisted of liposomal amphotericin B (LAmB) administered intrathecally (i.t.c.) at 0.006 mg/kg once weekly for 3 weeks, L-AmB administered intravenously (i.v.) at 10 mg/kg/day or voriconazole (VCZ) administered orally twice daily by gavage at 60 mg/kg/day. Combination therapies consisted of L-AmB i.t.c. + VCZ or L-AmB i.v. plus VCZ administered as in monotherapies. All treatments started 5 days after infection and continued until Day 19 post infection.
However, scarce data exist about their efficacy in animal models or in clinical practice. Administering drugs directly into the CNS could be an alternative, as occurs in other fungal infections that severely affect the CNS such as coccidioidomycosis, and especially in those cases where conventional therapy fails or a relapse of the infection occurs. In this study, the in vivo efficacy of intrathecal administration of L-AmB alone and in combination with VCZ was compared using a previously described murine model of cryptococcal meningitis caused by C. neoformans [11].
2. Materials and methods 2.1. Strain A clinical isolate of C. neoformans var. neoformans (serotype AD), strain FMR 8398, obtained from the cerebrospinal fluid of a human immunodeficiency virus (HIV)-positive patient, was used in the present study. The previously determined minimum inhibitory concentrations of AmB and VCZ for this strain were 0.5 g/mL and 0.03 g/mL, respectively [11]. The fungus was grown on Sabouraud dextrose agar (SDA) (Laboratorios Conda, S.A., Madrid, Spain) and was incubated at 35 ◦ C for 48 h.
2.2. Inoculum On the day of infection, a single colony was re-suspended in sterile saline and was adjusted to 11.5 × 103 yeasts/mL by dilution and haemocytometer counting. Viability was confirmed by placing 10-fold dilutions onto SDA plates and incubation for 5 days at 35 ◦ C, with the resulting number of colony-forming units (CFU) being 11.6 × 103 CFU/mL.
2.3. Infection model Four-week-old OF-1 male mice were anaesthetised by inhalation of fluothane vapours (0.5%). A total of 580 CFU/mouse [11] in a volume of 50 L was inoculated intracranially at the midline point on the cranium, 4–5 mm posterior to the eyes [12,13]. Mice were fully recovered from the procedure without neurological sequelae. Animal research procedures were approved by the Animal Welfare Committee of the Rovira i Virgili University (Reus, Spain).
2.4. Antifungal therapy Groups of 10 mice (5 per cage) were randomly assigned. All treatments began 5 days after infection and were continued for 15 days. Animals received the following monotherapies or combined therapies: L-AmB (AmBisome; Gilead Sciences Inc., Dublin, Ireland) at 10 mg/kg given intravenously (i.v.) into the lateral tail vein once daily; L-AmB at 0.006 mg/kg given intrathecally (i.t.c.) once a week for 3 weeks; VCZ (VFEND® ; Pfizer Inc., Madrid, Spain) at 30 mg/kg administered orally (p.o.) by gavage twice daily; L-AmB i.t.c. + VCZ p.o.; or L-AmB i.v. + VCZ p.o. The doses and schedule used in the combined therapies were the same as in the monotherapies. Control animals received a placebo of 50 L consisting of 0.9% saline administered i.t.c. once weekly for 3 weeks. From 1 day prior to the infection, mice receiving VCZ were given grapefruit juice (50% in water) instead of water [14]. Intrathecal administration of L-AmB was performed under anaesthesia induced by xylazine (10 mg/kg) and ketamine (100 mg/kg), both given intraperitoneally. A superficial skin incision was made over the lumbar vertebrae and the drug was delivered in a volume of 50 L into the subarachnoid space via lumbar puncture into the interspaces between the first and second lumbar vertebrae using a U-100 insulin syringe with a 27 G needle (Terumo Europe, Leuven, Belgium). After dosage, the skin incision was closed with wound clips. Mice were observed until they recovered from anaesthesia, at which time they showed no evidence of neurological abnormalities. Animals were checked twice daily and those showing signs of discomfort or survivors 21 days post infection were euthanised by CO2 anoxia. 2.5. Tissue burden At 48 h after the end of the therapy (i.e. Day 21 post infection), treated animals that survived were euthanised as described and their brains were removed. Owing to the quick death in untreated animals, euthanised animals from the control group were included on Days 11 and 12 post infection in the tissue burden study. Onehalf of the organ was weighed and mechanically homogenised in 0.9% saline. The homogenates were 10-fold diluted, placed onto SDA plates and incubated at 35 ◦ C for 72 h for CFU determination. 2.6. Histopathological studies The remaining half of the brain was placed in 10% buffered formalin. Samples were dehydrated, paraffin embedded, sliced into
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2.7. Statistical analysis Comparisons of survival were done by log-rank test and comparisons of fungal burden by Mann–Whitney U-test applying Bonferroni correction for multiple comparisons. All comparisons were done using GraphPad 4.0 for Windows (GraphPad Software Inc., La Jolla, CA). 3. Results 3.1. Survival
Fig. 2. Scattergram and median (horizontal bar) of colony-forming units (CFU) recovered from brains 21 days after intracranial infection with 580 CFU of Cryptococcus neoformans. Treatments consisted of liposomal amphotericin B (L-AmB) administered intrathecally (i.t.c.) at 0.006 mg/kg once weekly for 3 weeks, L-AmB administered intravenously (i.v.) at 10 mg/kg/day or voriconazole (VCZ) administered orally twice daily by gavage at 60 mg/kg/day. Combination therapies consisted of L-AmB i.t.c. + VCZ or L-AmB i.v. plus VCZ administered as in monotherapies. All treatments started 5 days after infection and continued until Day 19 post infection. Data from control group correspond to animals euthanised 11 days and 12 days post infection.
2 m sections and stained with haematoxylin and eosin, periodic acid-Schiff and Grocott’s methenamine silver stain. A pathologist blind to the treatment each animal had received carried out the microscopic examination of sections.
All control animals succumbed to the infection within 9–12 days post infection, but animals receiving monotherapy (VCZ, LAmB i.v. or L-AmB i.t.c.) or combination therapy (L-AmB i.v. + VCZ or L-AmB i.t.c. + VCZ) survived significantly longer (P ≤ 0.05) than control mice (Fig. 1). No differences were observed amongst therapies, with the exception of VCZ administered alone, which was significantly less effective in prolonging survival than the other treatments (P ≤ 0.012). 3.2. Fungal load The tissue burden study (Fig. 2) showed that control animals had a high fungal load in the brain (median log10 ± standard deviation 6.883 ± 0.306). VCZ administered alone failed to reduce tissue burden in comparison with the control group (P = 0.533), whilst LAmB administered i.t.c. or i.v. significantly reduced burdens with respect to the controls (P = 0.03 and 0.02, respectively). Combination of L-AmB i.t.c. or i.v. with VCZ showed a reduction in CFU counts compared with the untreated animals (P = 0.014 and 0.04, respectively), but the combination of L-AmB i.v. + VCZ did not show an advantage in reducing burdens over the monotherapy with
Fig. 3. Brain sections of mice infected intracranially with 580 colony-forming units (CFU)/animal. (A) Control animals showing enlargement of the meninges with massive inflammatory cells and encapsulated yeasts (arrows). (B) Animals treated with oral voriconazole (VCZ) showing enlargement of the meninges with abundant inflammatory cells, fungal elements and vascular congestion. (C) Animals treated with liposomal amphotericin B (L-AmB) intrathecally (i.t.c.) showing discrete enlargement of the meningeal structure and scarce yeasts. (D) Animals treated with L-AmB i.t.c. plus VCZ showing absence of fungal cells, discrete enlargement of the meninges and vascular congestion with oedema. Haematoxylin and eosin, ×250.
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L-AmB i.v. (P = 0.505). However, combination of L-AmB i.t.c. with VCZ resulted in a great reduction of fungal load in comparison with L-AmB i.t.c. alone (P = 0.039). This combination showed the greatest reduction in CFU counts, being significant in comparison with all the other therapies (P ≤ 0.039). 3.3. Histology findings The histopathological study showed extensive damage in the meningeal membranes of control animals. Macroscopically, they showed a mucous white-like layer covering the brain. Microscopically, control animals showed enlargement of the meninges with oedema and abundant inflammatory and meningeal reactive cells. In addition, numerous globose encapsulated fungal cells invading the meninges were also present (Fig. 3A). Despite the extensively damaged meninges, no cerebritis was observed and only scarce or no fungal cells were noticed in the brain parenchyma. Monotherapy-treated animals showed different degrees of meningitis with the presence of few fungal cells (Fig. 3B and C). In animals receiving L-AmB i.t.c. + VCZ, no fungal cells were observed and the meninges were apparently normal with the exception of small foci with evidence of tissue recovery with discrete oedema as a result of slight vascular congestion (Fig. 3D). 4. Discussion Meningitis is one of the most fatal complications of cryptococcosis in patients with acquired immune deficiency syndrome (AIDS) [15–17], although it has been also sporadically reported in non-HIV patients. These patients are frequently affected by the adverse side effects caused by administering antifungal treatments, especially with AmB and 5FC [18–20]. In cases of acute meningitis, trying to reduce the high burden in nervous tissue is crucial for improving the clinical outcome. Despite standard therapy, a growing number of reports have indicated that standard therapy is ineffective in some patients [3,21]. This has led to the need for more effective therapies for cryptococcosis affecting the CNS. VCZ has been suggested as an alternative therapy, and animal models of cryptococcal meningitis have demonstrated a reduction in fungal load and prolongation of survival after VCZ administration [22,23]. Although clinical and experimental data have shown VCZ as an alternative against cryptococcal meningitis, serum levels of this drug are highly variable amongst adults [24]. In the present study, the efficacy of L-AmB in achieving a fast reduction of CFU in CNS tissues was tested using an advanced acute model of meningitis in mice. Previous experimental studies conducted on rabbits have shown that continual intrathecal administration of lipidic AmB at concentrations ranging from 0.0015 mg/kg to 0.045 mg/kg did not show any signs or symptoms of neurological or toxic effects in the animals [25]. Although intrathecal administration is not the usual route of administration for AmB or its lipidic derivatives, this route is used in the treatment of coccidioidal meningitis in humans. Because coccidioidal meningitis cannot be cured with available antifungal agents, guidelines recommend therapy with AmB i.t.c. when consolidation therapy with azoles fails, at doses ranging from 0.01 mg to 1.5 mg and at intervals ranging from daily to weekly [26,27]. Few clinical studies have been conducted on the efficacy of intrathecal administration of antifungal agents against cryptococcal meningitis. However, in one of them, 22 non-HIV patients with meningitis caused by Cryptococcus were cured with AMB i.t.c. without any recurrence [28]. The current results demonstrate that L-AmB i.t.c. combined with VCZ significantly reduced the number of yeasts in the brain in comparison with the other therapies tested. In addition, histopathological findings showed that animals receiving such treatment had normal
meninges. The good distribution into the CNS of VCZ, which is not dependent on meningeal inflammation, is an additional advantage [10,29], making the combination of both drugs a potential alternative in salvage treatment of cryptococcal meningitis. Since CNS involvement has been documented in 52–61% of transplant recipients with cryptococcosis, and 25% of them have renal dysfunction at the time of diagnosis [17], the use of L-AmB i.t.c. at low doses could be considered a therapeutic approach in those patients, especially when systemic antifungal agents fail. Funding: This work was partially funded by the Universitat Rovira i Virgili (Reus, Spain). Competing interests: None declared. Ethical approval: Animal research procedures were approved by the Animal Welfare Committee of the Rovira i Virgili University (Reus, Spain).
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