Abstract
Superficial mycoses caused by Trichophyton rubrum are among the most common infections worldwide. T. rubrum infections are difficult to treat and are often associated with recurrences after interruption of the antifungal therapy. Nevertheless, reports on T. rubrum resistance to commonly used antifungal drugs are rare. In this study, we compared the in vitro resistance frequencies and development of resistance to terbinafine, itraconazole, amorolfine, and ciclopirox in T. rubrum. Results demonstrated that naturally occurring mutants were isolated at a frequency of 10−7 for itraconazole and 10−9 for terbinafine and amorolfine. To mimic conditions of body sites in which low drug levels are reached during therapy, T. rubrum was propagated for 10 transfers in media containing subinhibitory drug concentrations. Resistance to itraconazole, terbinafine, and amorolfine emerged at a higher frequency than was seen with spontaneous mutation. Itraconazole-resistant mutants also showed decreased susceptibility to amorolfine as well as to terbinafine, and amorolfine-resistant mutants were also less susceptible to terbinafine. No mutant resistant to ciclopirox was isolated, suggesting no propensity of T. rubrum to develop resistance to this drug. How different drug mechanisms of action can influence the onset of resistance is discussed.
INTRODUCTION
Dermatophytoses, the infections of keratinized tissues such as skin, hair, and nails by the highly specialized dermatophyte fungi, represent the most common type of human infection worldwide, particularly in aging, diabetic, or immunocompromised individuals (1–4). The main causative agent of dermatophytosis in developed countries is Trichophyton rubrum, an anthropophilic filamentous fungus that most commonly infects nails (onychomycosis) and skin (tinea pedis; tinea corporis) (1, 4–6). T. rubrum is known to account for almost 70% of all dermatophyte infections, and the incidence of infections due to this species has not changed in recent decades, although many efficient antifungal drugs have been introduced into the market during this period (4, 6, 7).
In clinical practice, several antimycotic agents are most frequently prescribed for the treatment of T. rubrum infections. Terbinafine (TRB) is a synthetic allylamine derivative that is widely used, both orally and topically, for treatment of dermatophyte onychomycosis and tinea (7). This drug is a potent inhibitor of the fungal squalene epoxidase, an enzyme involved in the biosynthesis of ergosterol (8). Azole drugs such as itraconazole (ITC) are fungistatic triazoles that block the ergosterol synthesis pathway by inhibiting the enzyme 14α-demetilase (9) and are popular for systemic and topical treatment of infections due to yeasts and dermatophytes (7). Amorolfine (AMF) is a member of the family of morpholine antimycotic agents that also block ergosterol synthesis (inhibition of sterol-Δ14-reductase and sterol-Δ7-Δ8-isomerase) and displays activity against a wide range of pathogenic fungi (8). Topical use of AMF in the form of nail lacquer is a common treatment for onychomycosis (10), and AMF-containing creams are used to treat skin dermatophytoses (11). Ciclopirox (CPX) is a hydroxypyridone which exhibits activity against nearly all the clinically relevant dermatophytes, yeasts, and molds. The main mechanism of action of CPX is its high affinity for trivalent cations (such as Fe3+), resulting in the inhibition of many metal-dependent enzymes that are responsible for the degradation of peroxides within the fungal cell (12). CPX and its olamine salt are available in multiple topical formulations, suitable for administration to the skin, nails, and vaginal mucosa (12).
T. rubrum infections and, in particular, onychomychosis are often intractable, and relapses occur frequently after cessation of the antifungal therapy (13, 14). One reason for unsuccessful antifungal management is poor adherence to long-term treatment regimens using topical antifungal drugs (7, 15). However, relapses following cessation of the antifungal therapy may also be due to T. rubrum acquisition of resistance, especially in the case of infections involving prolonged treatment with relatively low drug concentrations (14). Although rare, T. rubrum clinical isolates and in vitro-selected mutants resistant to ITC and TRB have already been described (14, 16–18).
The present in vitro study investigated the spontaneous occurrence of T. rubrum resistance to TRB, AMF, CPX, and ITC as well as the evolution of resistance to these drugs.
MATERIALS AND METHODS
Test organisms.
Three strains were used for this study: a reference T. rubrum strain (ATCC 28188) and two T. rubrum clinical isolates (CI-1 and CI-2). Strains CI-1 and CI-2 were isolated from clinical samples obtained at the Microbiology Unit of the Pisa University Hospital at the end of 2012 from different patients affected by onychomycosis. Patients declared that they were not undergoing topical or systemic antifungal therapy. This study was approved by the institutional review board. Strains were seeded on Sabouraud dextrose agar (SDA) and Dermasel agar plates and identified according to their microscopic and macroscopic features. T. rubrum ATCC MYA-4438 was included as a quality control isolate for broth dilution antifungal susceptibility testing as recommended by the CLSI (19). Strains were kept frozen at −70°C in Sabouraud dextrose broth (SDB; Becton, Dickinson) containing 30% glycerol. For each experiment, T. rubrum strains were taken from frozen stocks. Test organisms were routinely grown on SDA at 30°C.
Medium.
Assays were performed using RPMI 1640 medium with l-glutamine but without bicarbonate, buffered to pH 7.0 with 0.165 M 3-(N-morpholino)ethanesulfonic acid (Trek Diagnostic Systems Inc., Cleveland, OH).
Antifungal agents.
Stock solutions of TRB (Aminochemicals, Malta), ITC (Sigma-Aldrich, Switzerland), AMF (Sigma-Aldrich, Switzerland), and CPX (Erregierre, Italy) were obtained dissolving the drug powder (1 mg/ml) in 100% dimethyl sulfoxide (DMSO) (Sigma-Aldrich). Stock solutions were serially diluted using RPMI 1640 to yield the concentrations required by the experiments.
Inoculum.
T. rubrum was subcultured on SDA at 30°C for 7 to 15 days. All of the tested strains sporulated well after this period. Stock inoculum suspensions were obtained from each strain by covering the fungal colonies with sterile saline solution and gently rubbing the colonies with the tip of a transfer pipette. The resulting conidial suspensions were transferred to sterile tubes. Collected conidia were allowed to settle for 10 to 15 min, counted using a hemocytometer, and then adjusted to the required density by dilution in RPMI 1640. Plate counts were performed to verify the conidium concentrations by plating aliquots of the adjusted conidial suspensions on SDA.
MIC determination by the agar dilution assay.
To set up the experimental conditions to test both the natural and the induced resistance to drugs, minimum inhibitory concentration (MIC) determinations were performed at inocula of 108 CFU/plate and 105 CFU/plate by the agar dilution method described by Yamaguchi and coworkers (20), with some modifications. Serially diluted drug solutions (0.1 ml) were transferred into sterile petri dishes. Liquefied SDA medium (9.9 ml) was added at 45°C and immediately mixed. Control plates were prepared without addition of drugs. A 100-μl volume of the suspensions of dermatophytes was seeded by spot inoculation onto the control plates and on the media containing the antifungal agents. Plates were incubated at 35°C for 4 days. The antifungal concentration inhibiting growth was defined as the lowest concentration preventing growth of macroscopically visible colonies on drug-containing plates when visible growth was present on the control plates.
Natural resistance of T. rubrum to antifungals.
The natural resistance to antifungals was evaluated essentially as described by Osborne and colleagues (16). Briefly, stock inoculum suspensions of the dermatophytes were prepared to contain a total of about 109 CFU/ml. An aliquot of 0.1 ml of these suspensions was seeded on each SDA plate containing the antifungal agents at the concentrations inhibiting the growth of T. rubrum strains seeded at 108 CFU/plate. Colonies were counted after incubation at 30°C for 3 weeks. The resistance frequency to antifungals was calculated by dividing the number of CFU grown on the plates containing each drug by the total number of CFU spread on these plates. The colonies grown on the plates containing the antifungal agents were transferred to SDA plates containing 1- and 2-fold levels of the drug concentrations used for selection. Growth was checked after incubation for 1 week at 30°C.
Evolution of the antifungal drug resistance.
Strains ATCC 28188 and CI-1 were selected for evaluating the in vitro evolution of resistance to TRB, ITC, AMF, and CPX in T. rubrum. Conidia of each strain were serially propagated for 10 transfers (7 days of incubation for each transfer) in SDA containing a 0.5-fold level of the MIC of each drug. Experiments were performed essentially as previously described for Aspergillus fumigatus (21). In each transfer, 100 μl of a suspension containing about 106 conidia/ml was inoculated onto SDA plates containing the drug concentrations described above and the plates were incubated at 30°C. After the 5th and the 10th transfers, all conidia were collected and seeded on SDA plates containing 2-fold the MIC of each drug.
Antifungal susceptibility testing.
MICs were determined by the microtiter broth dilution method, as described for the CLSI M38-A2 standard methodology for the susceptibility testing of dermatophytes (19). Drug stocks were serially diluted in RPMI 1640 to yield twice the final strength required for the test and added (100 μl) to each well of 96-well microtiter plates (Greiner Bio-One, Frickenhausen, Germany). Inoculum T. rubrum suspensions were adjusted to 1 × 103 to 3 × 103 conidia/ml in RPMI 1640. Inocula (in 100-μl aliquots) were combined 1:1 with the test drug solutions in the microtiter plates to bring drug and inocula to the final desired test concentrations. Growth (RPMI 1640–1% DMSO) and sterility (RPMI 1640) controls were included for each tested isolate. Each organism was tested in duplicate, and the experiments were repeated three times. The inoculated plates were incubated at 30°C for 4 to 7 days. MICs were determined visually, and the MIC endpoint was defined as the lowest concentration that caused a reduction of ≥80% in fungal growth compared to the growth in the control well (drug-free medium). MIC differences of ±1 2-fold dilution were not considered significant.
Stability of drug-resistant mutants.
To ensure that resistant phenotypes were genetically stable in the absence of the drugs, selected mutants were serially propagated for three transfers (7 days incubation for each transfer) on nonselective SDA plates. After the third transfer, conidia were collected to determine the MIC by the microtiter broth dilution method, as described above.
RESULTS
Isolation of spontaneous antifungal-resistant T. rubrum mutants.
To isolate spontaneous mutants resistant to TRB, ITC, AMF, or CPX, a total of about 109 CFU of the ATCC 28188, CI-1, and CI-2 T. rubrum strains was plated on solid growth media containing the minimum inhibitory drug concentration determined by the agar diffusion assay for each antifungal agent. The highest incidence of resistant mutants was obtained with ITC, which selected mutants in the three strains with resistance frequencies ranging from 2.97 × 10−7 to 7.51 × 10−7 (Table 1). The frequencies of naturally occurring AMF-resistant derivatives for the three strains ranged from 1.14 × 10−9 to 5.79 × 10−9. Mutants resistant to TRB were obtained with strain ATCC 28188 (resistance frequency, 1.38 × 10−9) and strain CI-2 (resistance frequency, 2.89 × 10−9). No mutant resistant to CPX was obtained from any of the three T. rubrum strains. All colonies grown on the plates containing TRB or AMF and 10 randomly selected ITC-resistant colonies were able to grow when transferred to fresh plates containing 1- or 2-fold the amount of drug used for selection.
TABLE 1.
Spontaneous drug-resistant T. rubrum mutants obtained following direct selection on plates containing inhibitory drug concentrations
| Strain | Parametera | Value |
|||
|---|---|---|---|---|---|
| Terbinafine | Itraconazole | Amorolfine | Ciclopirox | ||
| ATCC 28188 | Total no. of CFU plated | 7.2 × 108 | 7.2 × 108 | 7.2 × 108 | 7.2 × 108 |
| No. of resistant colonies | 1 | 490 | 3 | 0 | |
| Resistance frequency | 1.4 × 10−9 | 6.8 × 10−7 | 4.1 × 10−9 | <1.4 × 10−9 | |
| CI-1 | Total no. of CFU plated | 8.7 × 108 | 8.7 × 108 | 8.7 × 108 | 8.7 × 108 |
| No. of resistant colonies | 0 | 654 | 1 | 0 | |
| Resistance frequency | <1.1 × 10−9 | 7.5 × 10−7 | 1.1 × 10−9 | <1.1 × 10−9 | |
| CI-2 | Total no. of CFU plated | 6.9 × 108 | 6.9 × 108 | 6.9 × 108 | 6.9 × 108 |
| No. of resistant colonies | 2 | 205 | 4 | 0 | |
| Resistance frequency | 2.9 × 10−9 | 3.0 × 10−7 | 5.8 × 10−9 | <1.4 × 10−9 | |
Resistance frequency data were calculated by dividing the number of CFU grown on the plates containing each drug by the total number of CFU spread on plates.
In vitro evolution of TRB, ITC, AMF, or CPX resistance in T. rubrum.
To analyze any in vitro evolution of resistance to TRB, ITC, AMF, or CPX, one clinical T. rubrum isolate (CI-1) and the reference ATCC 28188 strain were serially subcultured (10 transfers) on plates containing 0.5-fold the minimum drug concentration inhibiting growth on solid medium. Abundant fungal growth was obtained with both strains using these subinhibitory drug concentrations. After the 5th and the 10th transfer, conidia were collected and seeded on SDA plates containing 4-fold the drug concentrations used for transfers. After the 5th transfer, mutants with increased resistance to ITC were isolated from ATCC 28188 (resistance frequency, 9.6 × 10−5) and CI-1 (resistance frequency, 9.0 × 10−6) (Table 2). From ATCC 28188, mutants with increased resistance to TRB were also selected (resistance frequency, 1.3 × 10−7). After the 10th transfer, ATCC 28188 and CI-1 derivatives resistant to TRB, ITC, and AMF concentrations were isolated (see Table 2 for resistance frequencies). In the presence of subinhibitory drug concentrations, resistance to AMF, TRB, and ITC emerged rapidly in T. rubrum, with a frequency almost 100-fold higher than that of spontaneous drug resistance (Table 2). No CPX-resistant mutant emerged following exposure to subinhibitory CPX concentrations after 5 and 10 transfers.
TABLE 2.
In vitro evolution of drug resistance in T. rubrum following exposure to subinhibitory drug concentrations for 5 or 10 transfers before selection
| Strain | Transfer | Resistance frequencya |
|||
|---|---|---|---|---|---|
| Terbinafine | Itraconazole | Amorolfine | Ciclopirox | ||
| ATCC 28188 | 5th | 1.3 × 10−7 | 9.6 × 10−5 | <1.5 × 10−8 | <6.0 × 10−9 |
| 10th | 9.1 × 10−6 | 2.4 × 10−4 | 2.2 × 10−8 | <1.3 × 10−8 | |
| CI-1 | 5th | <5.2 × 10−8 | 9.0 × 10−6 | <3.4 × 10−8 | <1.7 × 10−8 |
| 10th | 4.4 × 10−7 | 2.6 × 10−5 | 9.1 × 10−8 | <1.2 × 10−8 | |
Resistance frequency data were calculated by dividing the number of CFU grown on the plates containing each drug by the total number of CFU spread on plates.
Antifungal resistance of selected mutants.
To evaluate the spectrum of resistance of the isolated mutants, MIC values of TRB, ITC, AMF, and CPX were determined by the broth microdilution assay. We analyzed all TRB- and AMF-resistant mutants and 5 randomly selected ITC-resistant mutants obtained from each parental strain following direct selection (naturally occurring mutants [S mutants]) or exposure to subinhibitory drug concentrations before selection (I mutants). The parental T. rubrum strains ATCC 28188, C-1, and CI-2 were assayed in parallel. For all these strains, the MICs of TRB, ITC, AMF, and CPX were 0.01 μg/ml, 0.08 μg/ml, 0.02 μg/ml, and 0.5 μg/ml, respectively. The results obtained with the quality control strain T. rubrum MYA-4438, which served as an internal control, were within the acceptable ranges recommended by the CLSI (i.e., MIC of CPX = 0.5 μg/ml).
Table 3 reports the fold increase in the MIC values of TRB, ITC, AMF, and CPX for the mutants compared to their parental strains. Mutants are clustered together on the basis of the drug used for selection, the parental strain from which they derived, and the method used for isolation (S or I mutants).
TABLE 3.
Fold increase in the MICs of terbinafine, itraconazole, amorolfine, and ciclopirox for drug-resistant T. rubrum mutants compared to their parental strainsa
| Mutant and drug susceptibility | Fold MIC increase |
|||
|---|---|---|---|---|
| Terbinafine | Itraconazole | Amorolfine | Ciclopirox | |
| ATCC 28188 | ||||
| TRB-S (n = 1) | 50 | ns | ns | ns |
| ITC-S (n = 5) | 4–8 | 4–8 | 4–8 | ns |
| AMF-S (n = 3) | 4–8 | ns | 16–32 | ns |
| TRB-I (n = 3) | 500–1,000 | ns | ns | ns |
| ITC-I (n = 5) | 8 | 4–8 | 32 | ns |
| AMF-I (n = 3) | 8–16 | ns | 64 | ns |
| CI-1 | ||||
| ITC-S (n = 5) | 4–8 | 4–8 | 4–8 | ns |
| AMF-S (n = 1) | 4 | ns | 16 | ns |
| TRB-I (n = 5) | 500 | ns | ns | ns |
| ITC-I (n = 5) | 8 | 4–8 | 32 | ns |
| AMF-I (n = 5) | 8–16 | ns | 32–64 | ns |
| CI-2 | ||||
| TRB-S (n = 2) | 500–1,000 | ns | ns | ns |
| ITC-S (n = 5) | 4–8 | 4–8 | 4–8 | ns |
| AMF-S (n = 4) | 4–8 | ns | 8–16 | ns |
Mutants were obtained following direct selection on plates containing inhibitory drug concentrations on the basis of selection (TBR, terbinafine; ITC, itraconazole; AMF, amorolfine) and the parental strain from which they derived. n, number of strains; ns, not significant (MIC differences of ±1 2-fold dilution were considered not significant).
A considerable increase in the MIC values of the antifungals that were used for selection was observed for the mutants compared to the parental strains. TRB-resistant mutants showed a 500- or 1,000-fold increase in the MIC values of TRB, ITC-resistant mutants a 4- or 8-fold increase in the MIC values of ITC, and AMF-resistant mutants 16- to 64-fold increases in the MIC values of AMF. Interestingly, the ITC-resistant mutants also showed increased MIC values of AMF (8- or 32-fold) and TRB (4- or 8-fold) and AMF-resistant mutants increased resistance to TRB (from 4- to 16-fold). No variation in the MICs of ITC and AMF was observed for TRB-resistant mutants. No substantial differences were observed in the MICs of resistant S and I mutants.
None of the mutants with increased resistance to ITC, AMF, or TRB showed altered MICs of CPX compared to the parental strains.
Analysis of the stability of the drug-resistant mutants following three transfers on nonselective medium revealed that all AMF-resistant mutants were genetically stable, while 5 of the 11 TRB-resistant mutants and 7 of the 25 ITC-resistant mutants exhibited restored susceptibility to TRB and ITC, respectively.
DISCUSSION
Routine antifungal susceptibility testing is not carried out in the case of isolation of dermatophytes from clinical samples. Therefore, it is difficult to define whether common relapses of dermatophytoses after the interruption of an antifungal therapy are due to poor compliance with the treatment or to infection with antifungal-resistant strains.
In this study, we aimed to evaluate the in vitro emergence of naturally occurring T. rubrum mutants resistant to TRB, ITC, AMF, and CPX and the evolution of resistance to these antifungals following exposure to subinhibitory drug concentrations.
The analysis of how frequently spontaneous drug-resistant T. rubrum mutants occur in vitro showed that the highest incidence of mutants is obtained with ITC (frequency of about 10−7). This result can be explained by the fact that fungistatic drugs, such as the triazoles, have the potential to leave more fungal survivors than fungicidal drugs (such as TRB, AMF, and CPX), and this larger effective population can contribute to a higher probability of resistance in the pathogen (21). A variety of biochemical and molecular mechanisms have been shown to contribute to drug resistance in eukaryotes (22). The resistance of dermatophytes to inhibiting agents involves the participation of target-enzyme modifiers and overexpression of ATP-binding cassette (ABC) transporters and stress-related proteins (23). In T. rubrum, two ABC transporters, TruMDR1 and TruMDR2, were identified as responsible for resistance to various antifungal drugs, such as ITC and TRB (24, 25). Candida albicans clinical isolates resistant to azole compounds and overexpressing the ABC transporter gene products CDR1 and CDR2 are less susceptible to the morpholine derivative AMF (26). Our finding that ITC-resistant mutants display increased MIC values of TRB and AMF (Table 3) suggests that ABC transporters play a role in the cross-resistance to these antifungals in T. rubrum. However, the observation that 28% of the ITC-resistant mutants exhibit restored susceptibility following three passages on nonselective medium suggests that other mechanisms can be involved in the development of ITC resistance in T. rubrum.
Spontaneous resistance to TRB occurred at a low frequency (about 10−9), in agreement with previous data reported by Osborne and coworkers (16). As already suggested, this low frequency of isolation appears to be compatible with resistance based on a single nonsilent nucleotide substitution in the gene encoding squalene epoxidase (27). Three results of the present study further support this hypothesis: (i) the high level of resistance to TRB displayed by TRB-resistant mutants (Table 3), (ii) the observation that TRB-resistant mutants do not display increased resistance to ITC and AMF (Table 3), which inhibit the activity of different enzymes in the ergosterol biosynthesis pathway (8, 9), and (iii) the restored susceptibility to TRB observed in 45% of the TRB-resistant mutants when drug pressure was removed.
To our knowledge, this is the first report describing the acquisition of resistance to AMF by T. rubrum. Spontaneous mutants resistant to AMF were isolated at a low frequency (about 10−9). In some yeasts, resistance to AMF has been associated with overexpression of ABC transporter genes and with cross-resistance to TRB (26, 28). Although the mechanisms that underlie resistance of dermatophytes to AMF are unknown, our finding that AMF-resistant T. rubrum mutants are also resistant to TRB suggests that drug efflux by ABC transporters can also contribute to drug resistance in this organism. The finding that AMF resistance is conserved after three passages in the absence of the drug further supports this hypothesis.
The emergence of drug resistance in all pathogenic microorganisms is an evolutionary process initiated by the exposure to antimicrobial agents. The emergence of resistance is a function of the rate of mutation to resistance and the size of the surviving population. T. rubrum acquisition of drug resistance during therapy may be responsible for relapses following cessation of the antifungal therapy, especially in the case of infections involving prolonged therapy with relatively low drug concentrations. The emergence of T. rubrum strains with greatly reduced susceptibility to TRB was previously reported in patients following prolonged therapy for onychomycosis (14). In order to investigate whether T. rubrum develops resistant mutants upon exposition to drugs in vitro, we grew T. rubrum in media containing subinhibitory concentrations of TRB, ITC, AMF, or CPX. In the presence of ITC, mutants with elevated resistance to ITC were isolated at a higher frequency than was seen with spontaneous mutation. A decreased susceptibility to ITC has already been described for T. rubrum subjected to sequential passages in the presence of the azole compound (17). Our results determined with TRB and AMF indicate that prolonged exposure to subinhibitory concentrations of these drugs also leads to significant loss of susceptibility (resistance frequency, about 10−7 to 10−8). It can be speculated, therefore, that T. rubrum can more easily develop resistance to ITC, TRB, and AMF at infection sites in which drug concentrations are not appropriately reached during therapy.
In this study, no difference was observed in the MICs of mutants obtained following direct selection (S mutants) or following exposure to subinhibitory drug concentrations before selection (I mutants). This result suggests that growth in the presence of subinhibitory drug concentrations, despite increasing the frequency of isolation of resistant strains, does not cause variations in the level of resistance compared to naturally occurring mutants.
CPX is a hydroxypyridone that is chemically and mechanically different from other antifungal agents, such as azole derivates, allylamines, and morpholines. Unlike those antifungal agents, CPX does not affect sterol biosynthesis and is not metabolized via cytochrome P450 (12). Interestingly, in the present study, no mutant resistant to CPX was obtained, thus suggesting that, under our experimental conditions, T. rubrum has no biochemical or molecular capacity to develop resistance to this drug, even after prolonged exposure to subinhibitory drug concentrations for several growth generations. Lack of tolerance of or resistance to ciclopirox in tests of this drug against C. albicans has previously been published (29). The multiplicity of the mechanisms of action of CPX may explain why there is no development of resistance to this drug, unlike the results seen with other antifungal drugs, and why this is the case not only in yeasts but also in dermatophytes.
In conclusion, the overall results obtained in this study indicate that spontaneous T. rubrum mutants resistant to AMF, TRB, and ITC can be isolated, although at low frequency, and that subinhibitory drug concentrations facilitate the emergence of resistant strains. In addition, our data indicate that, among the tested drugs, CPX is the only compound able to maintain the same efficacy against a wild-type population of T. rubrum and after prolonged use at subinhibitory concentrations. Its use in the topical therapy of dermatophytoses could contribute to reducing the relapse rate due to selection of drug-resistant strains.
ACKNOWLEDGMENT
This work was supported by Polichem S.A.
Footnotes
Published ahead of print 10 March 2014
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