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`Medical Mycology April 2004, 42, 159 /163
`
`Antifungal drug response in an in vitro model of
`dermatophyte nail infection
`
`C. S. OSBORNE*$, I. LEITNER*, B. FAVRE% & N. S. RYDER*$
`*Infectious Diseases Department, Novartis Research Institute, Vienna, Austria
`
`Despite terbinafine being fungicidal against Trichophyton rubrum in standard
`NCCLS assays and rapidly accumulating in nails in vivo, onychomycosis patients
`require prolonged terbinafine treatment to be cured. To investigate this, we
`developed a more clinically relevant onychomycosis in vitro test model. Human
`nail powder inoculated with T. rubrum and incubated in liquid RPMI 1640 salt
`medium, which did not support growth alone, developed extensive and invasive
`mycelial growth. Antifungal drugs were added at different concentrations and
`cultures incubated for 1 to 4 weeks. Fungal survival was determined by spreading
`cultures on PDA plates without drug and measuring CFU after 1 to 4 weeks
`incubation. Drug activity was expressed as
`the nail minimum fungicidal
`concentration (Nail-MFC) required for 99.9% elimination of viable fungus.
`Terbinafine Nail-MFC was 4 mg/ml after 1 week exposure, decreasing to 1 mg/ml
`after 4 weeks exposure, much higher than MFCs 5/0.03 mg/ml determined in
`standard NCCLS MIC assays. In contrast, other clinically used drugs were unable
`to kill T. rubrum after 4 weeks incubation in this model. Invasive mycelial growth
`on nail appears to protect T. rubrum from the cidal action of systemic drugs, thus
`providing a rationale for the long treatment periods in onychomycosis.
`
`Keywords
`
`antifungals, onychomycosis, Trichophyton rubrum
`
`Introduction
`
`is a common
`infection, onychomycosis,
`Fungal nail
`disease. Its prevalence can reach up to 8% in Nordic
`countries [1,2] and was even higher in a large scale
`study in North America [3]. Several heterogeneous
`factors also predispose some population groups to
`onychomycosis [2,4]. The main causative agents of
`nail infections are the dermatophytes, of which Tricho-
`phyton rubrum is the most frequently found [3,5]. These
`
`Received 19 August 2003; Accepted 24 November 2003
`Correspondence: Colin S. Osborne, Infectious Diseases Biology,
`Room 8654, Novartis Institutes for Biomedical Research Inc., 100
`Technology Square, Cambridge, MA 02139, USA. Tel.: +1 617 871
`3142; Fax: +1 617 871 7058; E-mail: colin.osborne@pharma.
`novartis.com
`$ Present address: Infectious Diseases Biology, Novartis Institutes
`for Biomedical Research Inc., 100 Technology Square, Cambridge,
`MA 02139, USA.
`% Present address: Department of Dermatology, University Hospital
`CHUV, 1011 Lausanne, Switzerland.
`
`– 2004 ISHAM
`
`fungi have a characteristic ability to utilise keratin as a
`nutrient source [6]. Terbinafine is highly effective in
`treating dermatophyte infections and acts by blocking
`ergosterol synthesis through the inhibition of squalene
`epoxidase, resulting in an accumulation of squalene,
`which is toxic to fungal cells [7]. MIC90 values for
`terbinafine, determined for T. rubrum strains according
`to standard NCCLS procedures, are 5/0.06 mg/ml [8 /
`11]. The fungicidal activity of
`terbinafine against
`dermatophytes is well established [12 /19], with mini-
`mum fungicidal concentrations (MFC) of 5/0.06 mg/ml
`against T. rubrum , and complete killing requiring not
`more than a few days [12,15,16,19].
`In vivo, terbinafine accumulates rapidly in human
`nail, where it persists for a long time [20,21]. For
`example, Faergemann et al.
`[21] reported that a
`maximum concentration of 0.39 mg/g is reached, and
`that levels of 0.09 mg/g are still present 55 days after the
`end of
`therapy. However, despite its potent cidal
`activity in vitro and its favourable pharmacokinetic
`properties in vivo, standard treatment of toenail infec-
`
`DOI: 10.1080/13693780310001656803
`
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`160 Osborne et al.
`
`tions with terbinafine requires 3 months of once-daily
`oral therapy [22,23]. Other therapies require similar or
`even longer periods of treatment [24].
`The purpose of this study was to develop a nail-
`based culture model
`for the testing of antifungal
`compounds. This model mimics the course of a natural
`nail fungal infection, with antifungal treatment starting
`only after mycelial growth is established. In this model,
`T. rubrum is much more resistant to the cidal action of
`terbinafine than in conventional assays.
`
`Materials and methods
`
`Trichophyton rubrum strains
`
`The five T. rubrum strains used in this study were
`clinical isolates from the Novartis Fungal Index (NFI)
`collection. The strains were from infected nails (NFI
`5132 and NFI 5143) or feet (NFI 5139 and NFI 5140),
`while NFI 5182 corresponds to the ATCC strain 18759,
`which caused dermatophytosis. To prepare stock in-
`ocula, cultures were grown on potato dextrose agar
`(PDA) (Merck, Whitehouse Station, USA) at 308C for
`1 /5 weeks. The conidia and mycelium was then
`harvested, dispersed in Sabouraud 2% dextrose broth
`(Merck), and stored at /808C after addition of 5%
`(vol/vol) dimethyl sulfoxide (DMSO) as cryoprotectant.
`Colony forming units (cfu) in these stock inocula was
`determined, after rapid thawing, by spreading on PDA
`plates 50 ml from 10-fold serial dilutions in a physio-
`logical saline solution, and counting the colonies after 1
`week’s incubation at 308C.
`
`Antifungal drugs
`
`Terbinafine, naftifine and itraconazole were synthe-
`sized at Novartis, Basel, Switzerland. Fluconazole was
`extracted and purified from commercial tablets of
`Diflucan (Pfizer) at Novartis, Vienna, Austria. Amor-
`olfine was from Roche Pharmaceuticals, Basel, Switzer-
`land. Clotrimazole, ciclopiroxolamine and griseofulvin
`were purchased from Sigma Chemical Co, St Louis,
`USA. All drugs were dissolved and two-fold serially
`diluted in dimethyl sulfoxide (DMSO).
`
`Determination of MIC and MFC
`
`MIC were determined in 96-well flat bottom assay
`plates with a slight modification of
`the NCCLS
`microdilution procedure M38-A [25,26]. The final
`concentration of DMSO was 1% and the size of the
`inoculum was 2.5/104 cfu/ml. Plates were incubated
`for 4 /5 days at 358C. Growth inhibition was scored
`visually with the aid of an inverted magnifying mirror
`
`from 4 to 0 according to NCCLS M38-A reference
`method [25]. The MIC corresponded to the lowest
`concentration giving a score of 1 (equivalent to about
`75% growth inhibition) and 100% MIC to the lowest
`concentration giving a score of 0 (equivalent to 100%
`growth inhibition). After MIC determination, starting
`from the last well in which growth was observed up to
`the highest drug concentration tested, 100 ml was
`transferred in duplicate onto potato dextrose agar
`(PDA) medium (Merck) in a 20 ml/9-cm-diameter
`plate. Plates were incubated for 1 week at 308C followed
`by visual inspection of growth. MFC corresponded to
`the lowest drug concentration (in the assay microtitre
`plate) at which no more than one colony subsequently
`grew in the 9-cm plate, corresponding to ]/99.9%
`killing activity.
`
`Preparation and collection of nail powder
`
`Human nail clippings were collected from healthy
`volunteers within the Novartis Research Institute.
`Finger and toe nails were pooled and ground to a
`powder using a stainless steel peppermill (Peugeot,
`Paris, France) followed by a nail micronizer (Delasco,
`Council Bluffs, USA). The nail powder was washed
`with chloroform and then ethanol, prior to being
`autoclaved at 1208C for 20 min.
`
`Nail model culture system and determination of nail MFC
`
`Autoclaved nail powder was added, at approximately
`10 mg per well, to 96-well plates. T. rubrum , 5 ml of 1/
`106 cfu/ml, was added directly onto the nail powder. A
`high cfu/low volume inoculate has been previously
`reported to give good uniform growth on nail frag-
`ments [27]. This was left at 358C for 1 h, after which 200
`ml RPMI select amine medium buffered with 0.165 mol/
`l MOPS (Invitrogen, San Diego, USA), which is
`essentially a balanced salts solution, were added (final
`cfu/ml: 2.5/104). The plate was returned to 358C for
`4 /5 days, by which time growth was well established. A
`small volume of drug (10 ml),
`initially dissolved in
`DMSO, and then in medium, was added to the desired
`concentration (final concentration of DMSO 1%) and
`the culture was returned to 358C. Drug incubation
`periods of 1, 2, 3 and 4 weeks were investigated. All
`experiments were performed in duplicate. After incuba-
`tion with drug, the content of each well was washed
`twice with RPMI 1640 (Invitrogen) before being
`transferred via an inoculating loop to a PDA plate
`(containing no drug)
`for nail MFC (Nail-MFC)
`determination. Plates were incubated at 308C and
`were examined and photographed after 1, 2, 3 and 4
`weeks. Nail minimum fungicidal concentration was
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`defined as the lowest drug concentration (in the assay
`microtitre plate) at which no more than two colonies
`subsequently grew in the 9-cm plate, corresponding to
`]/99.9% killing activity.
`
`Table 1 MIC, MFC, and Nail-MFC values of terbinafine against
`five Trichophyton rubrum strains
`
`Strain
`
`MICa
`
`100% MIC
`
`MFC
`(mg/ml)
`
`Nail-MFCb
`
`In vitro nail infection model 161
`
`Results
`
`Nail-MFC assay development with five T. rubrum strains
`
`In the presence of nail powder and RPMI select amine
`medium buffered with 0.165 mol/l MOPS, prominent
`mycelial growth of T. rubrum strains could be seen in
`each inoculated well after a few days of incubation at
`358C (data not shown). By lifting up the mycelium with
`forceps, it could be seen that the fungi were firmly
`attached to the nail particles. In control wells without
`nail powder the dermatophyte failed to grow (not
`shown), demonstrating that nail was an essential
`nutrient source in this assay. Attempts to quantitatively
`assess mycelium on the nail using fluorescent dyes
`failed because of the intrinsic, high fluorescence of nail
`(C. Osborne, unpublished data).
`To the mycelium grown on nail for 4 /5 days, various
`concentrations of terbinafine were then added and
`Nail-MFC determined as described in Materials and
`methods. Initially, 5 T. rubrum strains were tested with
`a drug incubation time of 1 week and an incubation
`time on PDA plates without drug of also 1 week. Nail-
`MFCs were very similar for all five strains tested.
`However, they were much higher than MFC values
`obtained by the standard NCCLS MIC assay (Table 1).
`In cases where fungal outgrowth occurred on the drug-
`free PDA plates, nascent fungal growth was observed to
`emanate directly from the nail pieces, suggesting a
`protective effect of the nail.
`
`Effect of incubation time on terbinafine activity
`
`In view of the similar results obtained with different
`strains of T. rubrum , subsequent experiments were
`performed with strain NFI 5143. A longer outgrowth
`incubation time of 4 weeks on drug-free PDA plates
`after the 1-week’s treatment with terbinafine slightly
`increased the Nail-MFC values from 2 to 4 mg/ml. This
`more stringent condition (4 weeks incubation on PDA
`plates) was employed for all subsequent experiments.
`The influence of incubation time with terbinafine on
`Nail-MFC was then investigated. The Nail-MFC for
`terbinafine decreased stepwise with longer exposure to
`the drug, dropping from 4 mg/ml after a 1-week
`exposure, to 1 mg/ml after a 4-week exposure (Fig. 1).
`After 2 and 3 weeks incubation with terbinafine the
`Nail-MFC was 2 mg/ml.
`– 2004 ISHAM, Medical Mycology, 42, 159 /163
`
`NFI 5132
`NFI 5139
`NFI 5140
`NFI 5143
`NFI 5182
`
`0.008
`0.008
`0.004
`0.008
`0.002
`
`0.016
`0.016
`0.008
`0.016
`0.004
`
`0.030
`0.030
`0.016
`0.030
`0.030
`
`2
`2
`2
`2
`1
`
`aMIC and 100% MIC correspond to drug concentrations required to
`fungal growth by 75% and 100% respectively. b1-week
`inhibit
`incubation with drug followed by 1-week’s incubation on PDA plates
`without drug.
`
`Activity of other antifungals
`
`In contrast to terbinafine, all of the other drugs tested
`(amorolfine, ciclopiroxolamine, clotrimazole, flucona-
`zole, griseofulvin,
`itraconazole, and naftifine) were
`unable to effectively kill T. rubrum growing on nail
`powder. In all cases, prominent fungal growth was
`observed on PDA plates at the highest concentrations
`tested (amorolfine 1 mg/ml, ciclopiroxolamine 128 mg/
`ml, clotrimazole 64 mg/ml,
`fluconazole 128 mg/ml,
`griseofulvin 64 mg/ml,
`itraconazole 4 mg/ml, and
`naftifine 8 mg/ml) even after a 4-week incubation period
`with these drugs.
`
`Discussion
`
`The standard NCCLS methodology for antifungal
`testing makes use of conidial suspensions [25,33], or
`as used by ourselves, a mixed suspension of mycelial
`fragments and conidia [26], growing in a rich medium.
`In contrast, the novel assay described in this report
`presents the test drug with the challenge of an
`established dermatophyte mycelium growing on and
`metabolising its natural substrate, the nail. We postu-
`late that this is much more akin to the clinical situation
`of onychomycosis in which the drug must overcome an
`established infection. Terbinafine Nail-MFCs obtained
`in this model, ]/1 mg/ml, were much higher than MFC
`values obtained after conventional MIC assays, 5/0.06
`mg/ml [12,15,16,19,26]. The cidal action of terbinafine
`was also much slower than in conventional assays,
`requiring 4 weeks incubation to achieve the lowest
`Nail-MFC value of 1 mg/ml. Nevertheless, terbinafine
`was the only drug among those tested that was able to
`eliminate viable T. rubrum from nail in this model.
`Terbinafine was previously reported by Seebacher [34]
`to have a much weaker killing activity against quiescent
`conidia-mycelium incubated in a physiological saline,
`versus growing mycelium incubated in Sabouraud
`broth. The presence of arthrospores, thought to be
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`Fig. 1 Influence of incubation time with terbinafine on Nail-MFC. Trichophyton rubrum NFI 5143, initially grown on nail without drug for 4
`days, was incubated for 1 week (A) or 4 weeks (B) with various concentrations of terbinafine: 4 mg/ml (a), 2 mg/ml (b), 1 mg/ml (c) and 0.5 mg/ml
`(d), and then transferred to PDA plates without drug and further incubated for 4 weeks.
`
`resistant to drug action [35], was not observed in our
`growth cultures. The combination of high Nail-MFC
`values and slow cidal action provides a feasible
`rationale for the 3-month treatment times in patients
`[22,23].
`The absence of any alternative nutrient supply in the
`culture medium means that the fungi must utilise the
`nails as their sole nutrition source by activating the
`secretion of keratinases [6,27]. Only a few previous
`studies have used nails as the only source of nutrition
`[28 /30]. Although, these systems have provided useful
`information, such as fungal morphological changes
`post drug exposure [28] or the effect of drugs on
`preventing nail plate invasion [30], none of them has
`adequately explained the long treatment time required
`to cure onychomycosis with systemic antifungals. Other
`models have used keratin particles from human stratum
`corneum as a substrate [31]. However, dermatophytes
`exhibit differences in their ability to degrade nails
`versus other types of keratin [32].
`In conclusion, the model described here provides an
`interesting and clinically more relevant tool for evalu-
`ating existing or novel antifungals for the treatment of
`onychomycosis. Future research directions with this
`model may include the study of additional nail patho-
`gens and rare terbinafine-resistant strains [36], use of
`
`drug combinations, and investigation of potential for
`development of drug resistance during nail infection.
`
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