Comparative ef cacies of commonly used disinfectants and antifungal pharmaceutical spray preparations against dermatophytic fungi

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1 ã Medical Mycology 2001, 39, 321±328 Accepted 20 February 2001 Comparative ef cacies of commonly used disinfectants and antifungal pharmaceutical spray preparations against dermatophytic fungi A. K. GUPTA*, I. AHMADy & R. C. SUMMERBELLz *Division of Dermatology, Department of Medicine, Sunnybrook and Women s College Health Sciences Center (Sunnybrook site), and the University of Toronto, Toronto, Ontario, Canada; ymedical Mycology, Laboratories Branch, Ontario Ministry of Health, Toronto, Ontario, Canada and Mediprobe Laboratories, Toronto, Ontario, Canada; zdepartment of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, Ontario, Canada and Centraalbureau voor Schimmelcultures, Utrecht, the Netherlands Introduction Arthroconidia from ve fungal strains belonging to three Trichophyton species, Trichophyton mentagrophytes, T. raubitschekii and T. tonsurans, were tested against commercial chemical disinfectants and pharmaceutical antifungal agents. The chemical disinfectants included chlorine, phenol, sodium dodecyl sulphate and several quaternary ammonium salts, while the two pharmaceutical preparations contained bifonazole and terbina ne as active agents. Arthroconidia were exposed to the antifungal agent either in a suspension solution for a given period of time and assayed for kill rate, or on a sprayed agar plate and monitored for surviving colonies over a period of 14 days. Chlorine (1%) and terbina ne (0 01%) were found to be high level disinfectants bringing about a rapid inactivation of conidia in all ve strains. Phenol was equally effective against T. raubitschekii and T. tonsurans; however, T. mentagrophytes cells were able to survive for up to 1 h in 5% phenol. Quaternary ammonium compounds were less rapid in their action against dermatophytes and were needed at a level of about 0 5% to be completely fungicidal. Three commercial spray formulations with a range of 0 1 to 0 3% quaternary ammonium salts were fungistatic against T. mentagrophytes strains. Bifonazole (1%) was also fungistatic in its action against T. mentagrophytes. Sodium dodecyl sulphate (0 5%) was largely ineffective against Trichophyton arthroconidia. Keywords One of the pressing concerns of patients recovering from dermatophytosis is the persistence of fomites in their personal belongings and living areas that expose them to re-infection. Surprisingly, little information is available on this subject, and our ability to disinfect dermatophyte inoculum from the human environment is restricted to material that either is treatable with stringent disinfectants such as chlorine or glutaraldehydes, or that can Correspondence: Aditya K. Gupta M.D., F.R.C.P.(C), 490 Wonderland Road South, Suite 6, London, Ontario, Canada, N6K 1L6. Tel.: ; fax: ; agupta@ execulink.com antifungal drugs, arthroconidia, chemical disinfectants, dermatophytes be discarded. Similarly, clinicians themselves require adequate means of instrument disinfection, especially in busy clinical practices where time constraints and large patient volume demand a quick and reliable method of disinfecting shared instruments between patients. Furthermore, at present our ability to suggest measures for outbreak control in public arenas is extremely limited. Traditional disinfecting techniques based on strong bleaching agents appear applicable for impervious surfaces such as oors, plumbing xtures and vents. However, these methods can not be recommended for soft materials such as footwear, hats and curtains that have been found to be fomites in many institutional outbreaks in recent years [1].

2 322 Gupta et al. Recently, several new disinfectants, many of them quaternary ammonium compounds, have appeared on the market claiming to disinfect dermatophyte inoculum. In general, these products are stated to have a broadbased antifungal activity; however, the evidence for their ef cacy against dermatophytes is lacking in the literature. In a 1993 USA study [2], all the Environment Protection Agency (EPA)-registered fungicidal commercial disinfectants except for glutaraldehydes failed the disinfection test against dermatophytic fungi (Trichophyton rubrum and T. mentagrophytes strains). There is, therefore, a need for exploring new strategies of disinfecting dermatophyte inoculum. A number of pharmaceutical products with high ef cacy against dermatophytes are now available in spray formulations. This raises the question as to whether these pharmaceutical spray formulations are employable for disinfecting patients belongings contaminated with infectious dermatophyte inoculum. Several recent studies have identi ed arthroconidia as the primary cause of infection in dermatophytes [3 5]. Many anthropophilic dermatophytes such as interdigital type T. mentagrophytes and T. tonsurans have been observed to produce arthroconidia in cultures [6]. Recently, these arthroconidia have been widely used as standardized infectious inoculum in studies on virulence mechanisms and new therapeutic agents [7]. There has been little success in producing in vitro arthroconidia in T. rubrum, which since its emergence after World War I, however, has become the most common etiological agent of human dermatophytoses in much of the world [8]. Research on this organism has lagged while the less prevalent T. mentagrophytes has been studied as a model system. The present study proposes the use of in vitro arthroconidia produced by strains identi ed as T. raubitschekii as a substitute for infectious cells of T. rubrum strains. We have recently determined culture conditions for obtaining arthroconidia from clinical strains of T. raubitschekii (Gupta et al., unpublished results). Prior to its description as a separate species in 1981 [9], T. raubitschekii was considered a variant of T. rubrum. Some taxonomists still consider T. raubitschekii to be within T. rubrum [10,11], and recent molecular studies have shown that it is identical to T. rubrum in key molecular features such as mitochondrial restriction fragment length polymorphism (RFLP) type and ribosomal internal transcribed spacer sequence [12]. Notwithstanding its ultimate placement, T. raubitschekii differs from T. rubrum in several phenotypic features, including the production of copious arthroconidia in vitro by many, if not all, recently isolated or well preserved strains (Gupta et al., unpublished results). The arthroconidia produced by T. raubitschekii strains, therefore, appear to offer a practical means of characterizing infective cells in members of the phylogenetic clade containing both these isolates and classically de ned T. rubrum. The current study assesses the antifungal ef cacy of several commonly used chemical disinfectants and two recently introduced pharmaceutical antifungal sprays against standardized arthroconidial inoculum of T. mentagrophytes, T. tonsurans and T. raubitschekii. All isolates used were recently obtained from human skin and nail infection. Materials and methods Fungal cultures Five abundantly arthroconidial Trichophyton isolates including two strains of T. mentagrophytes, two strains of T. raubitschekii and a single strain of T. tonsurans (Table 1) were obtained from clinical material at the Laboratories Branch of the Ontario Ministry of Health. Cultures were maintained at 28 o C by bimonthly transfer on Sabouraud CCG agar medium (10 g l 1 Bacto peptone, 40 g l 1 glucose, 15 g l 1 Difco agar, 50 mg l 1 chloramphenicol, 300 mg l 1 cycloheximide, 60 mg l 1 gentamicin, where the last three antibiotic additions de ne CCG). Arthroconidia were obtained from 3 4 week old fungal lawns grown on 80 ml Sabouraud CCG agar in 15 cm diameter plates. Preparation of arthroconidial inocula All steps were carried out under axenic conditions. Cultures with abundant arthroconidia on an 80 ml agar plate were scraped off gently with a heat sterile scalpel and suspended in 20 ml of phosphate-buffered saline (PBS), ph 7 2. In T. raubitschekii, areas of white, upraised, pasty texture on the colony surface, recognized microscopically as being composed of high concentrations of arthroconidia, were selectively utilized. Suspensions were examined microscopically and veri ed as containing a high proportion of arthroconidia, recog- Table 1 Species Fungal isolates Designated OMH* accession number T. mentagrophytes F T. mentagrophytes F T. raubitschekii FR T. raubitschekii FR T. tonsurans F Inoculum cell density (cfu ml 1 ) *, Ontario Ministry of Health dermatological specimen accession number from which the strain was derived.

3 Ef ciency of disinfectants and antifungal sprays 323 nized by characteristic globose morphology, relatively large size and conspicuous detachment scars on two sides of the conidium. The presence of minor proportions of microconidia in the preparations could not be entirely prevented, but was minimized by avoiding highly microconidial areas of the colony surfaces. Each suspension was agitated gently to produce a turbid suspension and the top 15 ml was gradually removed with a Pasteur pipette and ltered through a 5 ml plastic column padded with a porous disk. During this procedure any buildup of clumps and hyphal debris on the top of the disk was cleared with a metal loop. The ltrate was collected in a centrifuge tube and made up to 20 ml with PBS. For conidial count, the preparation was serially diluted in PBS and at each dilution triplicate 100 m l fractions were inoculated on 15 ml Sabouraud CCG agar in 8 5 cm diameter culture plates by spreading them evenly on the plate surface with a metal loop. The cell density of the inoculum was estimated at the dilution yielding about 100 individual colonies per culture plate. The cell densities of the ve fungi shown in Table 1 correspond to the 20 ml inoculum prepared for each strain. The disinfection studies were initiated within 3 h of conidial preparation. Disinfectants Suspension media formulations Solutions of 1% chlorine (sodium hypochlorite), 5% phenol, 0 5% benzalkonium chloride (BAC), 0 5% hexadecyltrimethyl ammonium bromide (cetrimide), 0 5% sodium dodecyl sulphate (SDS) and 0 01% terbina ne (Lamisil 1 ; Novartis Pharmaceutical Corporation, East Hanover, NJ, USA) were prepared by diluting 0 5 ml stock solutions in 4 5 ml autoclaved PBS, ph 7 2. For terbina ne, a powdered 250 mg tablet was extracted for 30 min in 2 5 ml dimethyl sulfoxide, centrifuged for 5 min at 1000 g and the clari ed extract was serially diluted to prepare a 0 1% stock solution in 80% ethanol. All other stock solutions were prepared in autoclaved distilled water using analytical grade chemicals. For the negative control, 0 5 ml of autoclaved water was added to 4 5 ml PBS. Spray formulations Aqueous solutions of 5% phenol, 0 5% BAC and 0 5% cetrimide prepared in autoclaved water were transferred to sterile 500 ml amber bottles tted with plastic spray assemblies to produce a 0 6 ml ne mist in each spray delivery. Lysol 1 domestic spray (Rickitt & Coleman Inc., Toronto, Ontario, Canada), Spray Nine* (Korkay System Canada Ltd., Gananoque, Ontario, Canada) and Brentdale germicidal spray (Brentdale Chemicals, Rexdale, Ontario, Canada) containing various quaternary ammonium compounds as active ingredients (Table 2) were obtained locally. Pharmaceutical preparations of 0 1% bifonazole spray (Mycospor 1 ; Bayer AG, Leverkusen, Germany) and 1% terbina ne hydrochloride (Lamisil 1 ; Novartis, Spartan, Kempton, South Africa) were obtained as physician samples. Antifungal evaluations The antifungal effect of each disinfectant was evaluated either by determining the kill rate after a given period of exposure to the disinfectant in a suspension solution, or by examining the viability of fungal cells on an agar plate sprayed with the disinfectant. In kill rate experiments, four replicate 5 ml suspension solutions containing 0 5 ml arthroconidial inocula were prepared for each disinfectant formulation, and 1 ml fractions were removed from each suspension at 0 25, 0 5, 1 and 24 h. The suspension was centrifuged immediately at 2000 g for 5 min, the supernatant solution was removed with a Pasteur pipette and the cell pellet was suspended into 10 ml PBS, ph 7 2. Triplicate 100 m l aliquots of each suspension were inoculated on Sabouraud slants in cm screw capped TB bottles by spreading cell suspensions evenly on agar surfaces with a metal loop. The slants were incubated horizontally at room temperature and examined twice a week for the emergence of new fungal colonies. Colony forming units (cfu) of each slant were determined as the maximum number of colonies recorded over a period of 2 weeks. A cfu count of 100 or more produced a dense lawn of fungal colonies over the 2 weeks growth period and was recorded as normal growth without inhibition in the present study. In spray treatment experiments, triplicate 100 m l aliquots of arthroconidial inoculum were evenly plated on Sabouraud agar medium in 8 5 cm diameter petri dishes and left Table 2 Trade name Commercial spray formulations Active chemical agents Lysol 1 0 1% n-alkyl (40% C 12, 50% C 14, 10% C 16 ) dimethyl benzyl ammonium chloride Spray Nine* 0 15% n-alkyl (5% C 12, 60% C 14, 30% C 1 6, 5% C 18 ) dimethyl benzyl ammonium chloride 0 15% n-alkyl (68% C 12, 32% C 14 ) dimethyl ethyl benzyl ammonium chloride Brentdale 0 1% n-alkyl (5% C 1 2, 60% C 1 4, 30% C 1 6, 5% C 18 ) dimethyl benzyl ammonium chloride 0 1% n-alkyl (68% C 1 2, 32% C 14 ) dimethyl ethyl benzyl ammonium chloride 1 6% tetrasodium ethylenediamine tetraacetate Mycospor 1 1% Bifonazole Lamisil 1 1% Terbina ne hydrochloride

4 324 Gupta et al. for 2 h at room temperature. Plates were treated with the given disinfectant by spraying a thin lm of about ml on the agar surface. The sprayed plates were incubated unsealed at room temperature. On day 3, a surface swab from of each plate was inoculated onto a Sabouraud slant. Both sprayed plates and swab test slants were examined regularly, and the numbers of newly emerging colonies were recorded for a period of 14 days. Any spray formulation allowing more than 300 fungal colonies on the sprayed agar plate was categorized as non-inhibitory. The spray was categorized as moderately inhibitory when the number of colonies on the plate ranged between 100 and 300. A fungistatic action was recorded when a large area of the sprayed plate was essentially free of fungal growth, while the swab test from the colony-free plate surface was positive for fungal growth. The spray was categorized as fungicidal when the sprayed plate was essentially free of fungal growth and the swab test was negative for growth. Results The test fungi listed in Table 1 were grown from dermatological specimens. They were selected on the basis at having primary cultures with abundant arthroconidia, which they were able to maintain in bimonthly subcultures throughout the course of this study. The predominance of arthroconidia in the fungal inoculum of each strain listed in Table 1 was con rmed microscopically. The ltered preparations were essentially free of hyphal fragments. In preliminary studies, the arthroconidial suspensions prepared in PBS were able to Table 3 Survival of T. mentagrophytes arthroconidia in suspension media commence germination within 30 h at room temperature, and were practically 100% viable. The centrifugation at 2000 g for 5 min employed in this study for cell washing had little effect on the arthroconidium germination rate (results not shown). In suspension experiments, the arthroconidia of the ve fungal strains remained completely viable during the entire period of 24 h incubation in PBS solutions added with water as negative disinfectant control (Tables 3 5). A colony number of over 100 in each 16 ml TB bottle completely covered the entire surface of a 10 ml agar slant that for practical purposes was noted as normal growth in the absence of a disinfectant effect. Similarly, in spray experiments, a colony count of more than 300 on water-sprayed 15 ml agar in 8 5 cm diameter plates (Tables 6 8) covered the entire agar surface under these conditions. Of the three species tested, T. mentagrophytes showed the highest ability to survive exposures to chemical disinfectants (Tables 3 8). Both strains of T. mentagrophytes were able to survive exposures to phenol, BAC, cetrimide and SDS. With phenol in the suspension, the survival rate of T. mentagrophytes arthroconidia was low and occurred only when exposure was less than 1 h. With SDS in the suspension, T. mentagrophytes strains remained essentially viable over the given period of 24 h. With the two quaternary ammonium compounds, BAC and cetrimide, both strains of T. mentagrophytes survived well for up to 1 h and even after 24 h some ability to survive was observed in both strains. The best disinfectants against T. mentagrophytes strains appear to be chlorine and terbina ne. An exposure of 15 min to either of these two agents was suf cient to kill all cells of the two strains (Table 3). A cfu after exposure time (h)* Treatment Isolate Water F >100 >100 >100 >100 F >100 >100 >100 >100 1% Chlorine F F % Phenol F F % BAC F > F > % Cetrimide F >100 > F >100 > % SDS F >100 >100 >100 >100 F >100 >100 > % Terbina ne F F *, Values are averages of three agar slants standard deviation.

5 Ef ciency of disinfectants and antifungal sprays 325 Table 4 Survival T. raubitschekii arthroconidia in suspension media 15-min exposure to chlorine and terbina ne was also suf cient to kill T. raubitschekii and T. tonsurans (Tables 4 and 5). Phenol was similarly rapid in killing all cells of these two species. With BAC, one strain of T. raubitschekii did survive a 15-min exposure while 30 min exposure to either BAC or cetrimide was completely lethal to both strains (Table 4). Similarly, T. tonsurans was killed by a 15 min exposure to BAC and 30 min exposure to cetrimide (Table 5). With SDS both T. raubitschekii and T. tonsurans survived well, particularly during the rst hour of 24 h of exposure to this agent (Tables 4 and 5). T. mentagrophytes also showed a higher ability to survive disinfectant sprays than the other two species. Of the nine antifungal sprays employed in these experiments, only phenol, BAC and Lamisil 1 were clearly fungicidal to both strains of this species (Table 6). The other six spray formulations were essentially fungistatic cfu after exposure time (h)* Treatment Isolate Water FR-54 >100 >100 >100 >100 FR-3393 >100 >100 >100 >100 1% Chlorine FR FR % Phenol FR FR % BAC FR FR % Cetrimide FR FR % SDS FR FR % Terbina ne FR FR *, Values are averages of three agar slants standard deviation. Table 5 Survival of T. tonsurans F arthroconidia in suspension media cfu after exposure time (h)* Treatment Water >100 >100 >100 >100 1% Chlorine % Phenol % BAC % Cetrimide % SDS >100 > % Terbina ne *, Values are averages of three agar slants standard deviation. in their action as subcultures, from apparently growth free areas of the sprayed plates, yielded good growth of fungal colonies after their transfer to spray free agar media indicating only a suppression of T. mentagrophytes conidia by the spray. In addition, a number of colonies of T. mentagrophytes were formed on the plates sprayed with cetrimide, SDS, Lysol 1 and Brentdale indicating a high level of tolerance in some cells to the presence of these disinfectants (Table 6). T. raubitschekii and T. tonsurans, on the other hand, showed no growth on plates sprayed with antifungal agents (Tables 7 and 8). However, a fungistatic effect of SDS was observed in both species. The effect of all other antifungal agents was fungicidal against T. raubitschekii and T. tonsurans strains (Tables 7 and 8). Discussion Given that arthroconidia constitute the primary means of transmitting Trichophyton infections in humans and animals, the present study offers a direct means of examining the potential effectiveness of chemical disinfectants against dermatological fomites in clothing, in patients living environs, and on public surfaces. We have been able to obtain dense arthroconidial inocula from the ve selected Trichophyton clinical isolates throughout the course of this study. Although the microscopic examination of these inocula usually showed the presence of other conidia, particularly a fairly numerous microconidia in T. tonsurans and one of the T. raubitschekii isolates, the arthroconidia were greatly predominant component in all the preparations. Arthroconidia are considered to be the most resistant of

6 326 Gupta et al. Table 6 Survival of T. mentagrophytes arthroconidia on sprayed agar plates Spray Isolate # F Isolate # F Trichophyton conidia [1,4]; therefore, the presence of other less resistant conidia is unlikely to affect our disinfectant ef cacy results. Moreover, the isolates showing some microconidia in the inoculum were among the least resistant isolates in disinfection studies. The results presented in this study, therefore, provide a reliable measure of the antifungal ef cacy of given chemical agents against infectious cells of Trichophyton strains. The antifungal agents used in the present study included two of the oldest established [13] chemical disinfectants, chlorine (bleach) and phenol, several recently developed quaternary ammonium compound complexes, a sodium dodecyl sulphate preparation and two currently available pharmaceutical antifungal sprays, Lamisil 1 and Mycospor 1. The exposure of arthroconidia to these chemicals was monitored either in a suspension solution for a xed period of time [14] or on a sprayed agar plate covered with an unsealed lid. These two modes of disinfectant applications were selected to correspond, respectively, rst to the disinfection of instruments and objects in chemical baths and the cfu* Classi cation of action cfu* Classi cation of action Water >300 None >300 None 5% Phenol 0 Fungicidal 0 Fungicidal 0 5% BAC 0 Fungicidal 0 Fungicidal 0 5% Cetrimide 3 1 Fungicidal 6 3 Fungicidal 0 5% SDS 12 4 Fungistatic 7 3 Fungistatic Lysol 1 0 Fungistatic 4 1 Fungistatic Spray Nine 0 Fungistatic 0 Fungistatic Brentdale 3 1 Fungistatic 5 2 Fungistatic Mycospor 1 0 Fungistatic 0 Fungistatic Lamisil 1 0 Fungicidal 0 Fungicidal *, Values are averages of three agar plates standard deviation. Table 7 Survival of T. raubitschekii arthroconidia on sprayed agar plates Spray Isolate # FR-54 Isolate # FR-3393 second to the disinfection by spray of exposed hard and soft surfaces. These two modes of disinfection differ both in the amount of the chemical present and the duration of contact made with the organism. The suspension experiments created a contact between the fungal conidia and the disinfectant that could be de ned both in terms of time and concentration. The spray experiments, on the other hand, resulted in an inde nite exposure of conidia to a varying amounts of disinfectant on the agar plate left after the evaporation of liquid from the sprayed surface. In our suspension experiments, the contact between conidia and disinfectant was brought to an end by a centrifugal cell wash in PBS solution. In our preliminary study of phenol and terbina ne against T. mentagrophytes, the kill rates estimated after the rst cell wash in PBS remained unchanged when cells were cleaned further by several consecutive washes in the buffer. Similarly, after the rst wash in PBS, a tenfold dilution of conidial suspension resulted in about a tenfold decrease in colony count, thus precluding a signi cant inhibition by the residual disinfectant left in the washed suspension. These results indicate the cfu* Classi cation of action cfu* Classi cation of action Water >300 None >300 None 0 5 % BAC 0 Fungicidal 0 Fungicidal 0 5% Cetrimide 0 Fungicidal 0 Fungicidal 0 5% SDS 0 Fungistatic 0 Fungistatic Lysol 1 0 Fungicidal 0 Fungicidal Spray Nine* 0 Fungicidal 0 Fungicidal Brentdale 0 Fungicidal 0 Fungicidal Mycospor 1 0 Fungicidal 0 Fungicidal Lamisil 1 0 Fungicidal 0 Fungicidal *, Values are averages of three agar plates.

7 Ef ciency of disinfectants and antifungal sprays 327 Table 8 Survival of T. tonsurans F arthroconidia on sprayed agar plates cfu* adequacy of the washing procedure used in the present study. In other studies, a special washing formulation called Dey-Engley medium [2] has been used to neutralize disinfectants in suspension solutions. This medium is, however, rich in organic nitrogen, and the presence of amino acids and proteins is known to inhibit growth in certain Trichophyton species [15]. In our study, a wash in PBS provided an effective mean of terminating the fungal exposure to the disinfectant solution. Of the various groups of disinfectant examined in the present study, chlorine and terbina ne exhibited by far the strongest fungicidal action against Trichophyton inoculum and can be classi ed as high level disinfectants for dermatophytes. Recently, the standard procedure for surface disinfection using 0 4% chlorine for 2 min has been shown to be inadequate for killing fungal conidia on cereal grains [16]. The data obtained in the present study show that a 15 min exposure to 1% chlorine is suf cient to kill a highly dense inoculum of Trichophyton conidia. Chlorine is, however, highly corrosive and causes irritation in closed surroundings making it of limited use in many areas. Phenol is another broadspectrum disinfectant; however, because it is usually effective at a concentration that can potentially be toxic to humans, its use has declined rapidly in recent years [12]. Our study shows that even at 5% concentration it is not completely effective against T. mentagrophytes. The other two species were found to be resistant to about 3% phenol (results not shown). SDS, a protein denaturant with high antiviral activity [17], was ineffective against all dermatophytic species tested in the present study. A number of quaternary ammonium salts used in our present study exhibited a relatively low-level antifungal activity. In suspension experiments, both BAC and cetrimide were able to kill T. raubitschekii and T. tonsurans in about 30 min, but a number of T. mentagrophytes cells were able to survive even after 24 h exposure to these disinfectants. Both BAC and Classi cation of action Water >300 None 5% Phenol 0 Fungicidal 0 5% BAC 0 Fungicidal 0 5% Cetrimide 0 Fungicidal 0 5% SDS 0 Fungistatic Lysol 1 0 Fungicidal Spray Nine* 0 Fungicidal Brentdale 0 Fungicidal Mycospor 1 0 Fungicidal Lamisil 1 0 Fungicidal *, Values are averages of three agar plates. cetrimide were able to kill all T. mentagrophytes cells when deposited on the agar plate in spray experiments, suggesting that an exposure period longer than 24 h is required with these disinfectants to complete the fungicidal effect. Interestingly, the quaternary ammonium compound formulations of the three commercial disinfectant sprays used in the present study produced only a fungistatic effect against T. mentagrophytes. It may be noted here that our spray formulations of BAC and cetrimide contained 0 5% (w/v) of these disinfectant, whereas the total amount of quaternary ammonium compound in the three commercial disinfectants varied between 0 1 and 0 3%. It appears that a concentration of about 0 5% quaternary ammonium compounds may be required in spray formulations to ensure a complete inactivation of Trichophyton species. Mycospor 1 spray also produced a fungistatic effect against T. mentagrophytes. Mycospor 1 has a high concentration (1% w/v) of bifonazole and it was not therefore considered necessary to increase its concentration further to determine the potentially fungicidal dose of this drug for T. mentagrophytes. Mycospor 1 was fungicidal against T. raubitschekii and T. tonsurans. Terbina ne was markedly fungicidal against all Trichophyton strains studied here. In our suspension experiments, a terbina ne concentration of 0 01% was formulated since a similar level of terbina ne has been used by other workers to screen resistant cells of pathogenic yeasts [18]. All ve strains of the three Trichophyton species studied here were killed within 15 min of exposure to 0 01% terbina ne. In our other experiments, a 100% kill rate was maintained against the ve strains by a terbina ne concentration as low as % (data not shown) indicating a markedly high sensitivity of Trichophyton species against this drug. The spray formulation in Lamisil 1 contains 1% terbina ne and it was evidently fungicidal against all strains studied here. It appears from our results that a much lower concentration of terbina ne will be suf cient to produce a fungicidal spray against Trichophyton conidia. This striking antifungal ef cacy of terbina ne against Trichophyton conidia appears to make this drug a strong candidate for disinfection of various fomites potentially involved in contagion and re-infection. Currently, our laboratory has been studying the antifungal activity of terbina ne against non-dermatophytic agents of onychomycosis. That T. mentagrophytes is more resistant to chemical disinfectants than other species of Trichophyton is consistent with other data available in the literature. The work of Terlecky & Axler [2] shows a considerable tolerance of T. mentagrophytes strains against various chemical disinfectants. Although, their study showed a

8 328 Gupta et al. high level of variability among T. mentagrophytes in resisting disinfectants, the isolates of T. mentagrophytes used in our study were only marginally different from each other. It appears that F and F used in our study are similar to the highly resistant T. mentagrophytes isolate FD studied by Terleckyj & Axler [2]. These isolates appear to be good candidates for use as test organisms in studying the ef cacy of antifungal agents against dermatophytes. It may be noted here that while Spray Nine* is stated to pass the EPA fungicidal test in which anthropophilic T. mentagrophytes var. interdigitale is exposed to the agent, its action against strains F and F used in the present study was fungistatic in nature. Both F and F were isolated from humans and are morphologically consistent with anthropophilic strains of T. mentagrophytes, they are therefore suitably representative of human-infecting isolates, for use as highly resistant test organisms in the disinfection protocols. In conclusion, the striking ef cacy of the well-studied terbina ne offers a potentially new source of noncorrosive, non-toxic disinfectant against dermatophytes. Acknowledgements We thank Sal Albreish and his staff for supplying arthroconidial Trichophyton cultures. References 1 Rippon JW. Dermatophytoses. In: Medical Mycology: the Pathogenic Fungi and the Pathogenic Actinomycetes, 3rd edn. Philadelphia: Saunders, 1988: Terleckyj MS, Axler DA. Ef cacy of disinfectants against fungi isolated from skin and nail infection. J Am Pod Med Assoc 1993; 83: Miyazi M, Nishimura K. Studies on arthrospore of Trichophyton rubrum (I). Japan J Med Mycol 1971; 12: Emyanitoff RG, Hashimoto T. The effects of temperature, incubation atmosphere, and the medium composition on arthrospore formation in the fungus Trichophyton mentagrophytes. Can J Microbiol 1978; 25: Aljabre SHM, Richardson MD, Scott EM, et al. Germination of Trichophyton mentagrophytes on human stratum corneum in vitro. J Med Vet Mycol 1992; 30: Summerbell RC, Kane J. The genera Trichophyton and Epidermophyton. In: Kane J, Summerbell RC, Sigler L, Krajden S, Land G, eds. Laboratory Handbook of Dermatophytes. Belmont: Star Publishing Company, 1997: Rashid A, Edward M, Richardson MD. Activity of terbina ne on Trichophyton mentagrophytes in a human living skin equivalent model. J Med Vet Mycol 1995; 33: Summerbell RC, Li A, Haugland R. What constitutes a functional species in the asexual dermatophytes? Microbiol Cult Coll 1997; 13: Kane J, Salkin IF, Weitzman I, et al. Trichophyton raubitschekii sp. nov. Mycotaxon 1981; 13: Matsumato T, Ajello L. Current taxonomic concepts pertaining to the dermatophytes and related fungi. Int J Dermatol 1987; 8: Ishizaki H. Fungal taxonomy based on mitochondrial DNA analysis. Jpn J Med Mycol 1993; 34: Summerbell RC, Haugland RA, Li A, Gupta AK. trna gene internal transcribed spacer 1 and 2 sequence of asexual, anthropophilic dermatophytes related to Trichophyton rubrum. J Clin Microbiol 1999; 37: Jeffrey DJ. Chemicals used as disinfectants: active ingredients and enhancing additives. Rev Sci Tech Off Int Epiz 1995; 14: Ohta S, Makino M, Nagai K, et al. Comparative fungicidal activity of a new quaternary ammonium salt N-alkyl-N-2- hydroxy-ethyl-n,n-dimethylammonium butyl phosphate and commonly used disinfectants. Biol Pharm Bull 1996; 19: Kane J. The biological aspects of the Kane/Fischer system for identi cation of dermatophytes. In: Kane J, Summerbell RC, Sigler L, Krajden S, Land G, eds. Laboratory Handbook of Dermatophytes. Belmont: Star Publishing Company, 1997: Andrews S, Pardoel D, Harun A, et al. Chlorine inactivation of fungal spores on cereal grains. Int J Food Microbiol 1997; 35: Howett MK, Neely EB, Christensen ND, et al. A broadspectrum microbiocide with virucidal activity against sexually transmitted viruses. Antimicrob Agents Chemother 1999; 43: Ryder NS, Wagner S, Leitner I. In vitro activities of terbina ne against cutaneous isolates of Candida albicans and other pathogenic yeasts. Antimicrob Agents Chemother 1998; 42: