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Isolation and characterization of the antifungal resistance genes of trichophyton mentagrophytes
P45
P47
Cidofovir diphosphate inhibits molluscum contagiosum DNA polymerase activity Takahiro Watanabe, MD, Department of Dermatology, University of Tokyo, Tokyo, Japan; Tamaki Kunihiko, MD, Department of Dermatology, University of Tokyo, Tokyo, Japan
A double-blind, randomized controlled trial of duct tape for the treatment of common warts in an adult population Rachel Wenner, MD, VA Medical Center, Minneapolis, MN, United States; Sharone Askari, MD, VA Medical Center, Minneapolis, MN, United States; Peter Cham, MD, VA Medical Center, Minneapolis, MN, United States; Erin Warshaw, MD, MS, VA Medical Center, Minneapolis, MN, United States Objective: To evaluate the efficacy of duct tape occlusion therapy for the treatment of common warts.
Molluscum contagiosum (MC) produces a benign papular eruption of multiple umbilicated cutaneous tumors. In HIV-infected individuals, extensive and recalcitrant MC lesions occur that represents a therapeutic challenge. Several studies describe the successful use of cidofovir, a nucleoside analog of deoxycytidine monophosphate, applied topically or by intravenous infusion for otherwise recalcitrant MC. The purpose of this study was to examine the effect of nucleoside analogs on the enzymatic activity. Because molluscum contagiosum virus (MCV) cannot be grown in tissue culture cells and does not infect animals, we have expressed MCV DNA polymerase gene by in vitro transcription-translation. DNA polymerase activity was measured by the incorporation of radioactive deoxycytidine into activated DNA. Twenty to fifty micromolars of CDV diphosphate, an active intracellular metabolite of CDV, was found to inhibit in vitro translated MCV polymerase activity by 50%. CDV diphosphate elicited a similar inhibitory effect on DNA polymerase of herpes simplex virus type 1 and cowpox virus. We believe these results provide for the first time biochemical support for a therapeutic use of cidofovir in the treatment of MC. Commercial support: None identified.
Design: A double-blinded, controlled, clinical intervention trial. Setting: Veterans Affairs Medical Center. Participants: A total of 90 immunocompetent adult volunteers with at least one wart measuring between 2 mm and 15 mm were enrolled in this study between from October 2004 until July 2005; 78 patients completed the study. Intervention: Patients were randomized using a computer-generated code to receive either pads consisting of moleskin with transparent duct tape (active) or moleskin alone (control). Patients were instructed to wear the pads for 7 consecutive days and leave the pad off on the seventh evening. This process was repeated for 2 months or until the wart resolved, whichever occurred first. Follow-up visits occurred at 1 and 2 months. Main Outcome Measure: One hundred percent resolution of the wart. Secondary outcomes included change in size of the target wart and recurrence rates at 6 months for warts with complete resolution. Results: There were no statistically significant differences in the proportions of patients with resolution of the target wart (duct tape 5 8/40 (20%) vs. control 5 9/41 (22%)). Of patients with complete resolution, 75% (6/8) in the duct tape group and 33% (3/9) of those in the control group had recurrence of the target wart by sixth months. Conclusions: Occlusive therapy with transparent duct tape is not significantly better than moleskin alone for treatment of common warts in adults. Limitations: Limitations include the use of an acrylic-based adhesive transparent duct tape. Investigator-initiated study was supported by a Minnesota Medical Foundation Medical Student Grant (non-profit foundation).
P46
P48
Isolation and characterization of the antifungal resistance genes of trichophyton mentagrophytes Aditya Gupta, MD, PhD, Mediprobe Research, Inc, London, Ontario, Canada; Jagpal Singh, PhD, Mediprobe Research, Inc, London, Ontario, Canada; Muhammad Zaman, PhD, Mediprobe Research, Inc, London, Ontario, Canada Trichophyton rubrum and T mentagrophytes are the major fungal pathogens of onychomycosis, a major dermatophytic skin disorder present in 6% to 13% of the North American population resulting in nail plate thickening and discoloration. Previous observations in the literature suggest that T rubrum can acquire increased antifungal resistance, while direct evidence of T rubrum resistance to tioconazole as well as cross-resistance to acridine and ethidium bromide upon increasing exposure to the azole derivative have been reported in vitro. There are also reports that T rubrum can acquire resistance to terbinafine and other squalene epoxidase inhibitors in vitro. The observation that newer and more potent antifungal drugs, such as terbinafine, exhibit a mycological cure rate of 60% to 80% in patients along relapse rates approaching 10% or higher, indirectly suggest that T rubrum drug resistant strains may play an important role in these phenomena. Despite these reports, the molecular mechanisms of T rubrum and T mentagrophytes drug resistance are poorly understood, and a clear understanding of this process is essential for designing new antifungal drugs and for formulating new strategies for effective therapy. Membrane transporters with ATP-binding cassette motifs (ABC transporters) are known to be responsible for conferring multiple drug resistance phenotypes in a wide range of organisms. Our research focuses on the isolation and characterization of ABC transporter genes in T rubrum and T mentagrophytes and investigating their possible involvement in conferring antifungal resistance. First, trichophyton cell lines with reduced fluconazole susceptibility were selected by successive passages in media supplemented with increasing fluconazole concentrations. PolyA1 RNA extracted from these cell lines were then used to clone a cDNA library. Screening of this library using primers specific for human, fungal and bacterial ABC transporter motifs resulted isolation of a single 4kB candidate mdr gene from T mentagrophytes, named Tmmdr1. We are currently completing sequencing of Tmmdr1 to fully confirm its homology to other mdr genes. Functional characterization of Tmmdr1 will be undertaken by transforming and expressing the gene in drug-sensitive Saccaromyces cerevisae strains to determine if it can reverse drug sensitivity. The use of this yeast as an assay organism to select drug-resistant genes is well documented from a wide variety of prokaryotic and eukaryotic species.
Comparative trial of topical application of silver sulphadiazine 1% 1 cerium nitrate 0.4% and acyclovir in labial herpes simplex lesions Omar Lupi, MD, MMSc, PhD, Hospital Universitario Clementino Fraga Filho/UFRJ, Rio de Janeiro/RJ, Brazil; Mariana Gusm~ao, MD, Universidade Federal do Rio de Janeiro, Rio de Janeiro/RJ, Brazil; Ange´lica Gonc¸alves, MD, Universidade Federal do Rio de Janeiro, Rio de Janeiro/RJ, Brazil; Paula Dadalti, MD, MMSc, PhD, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil Introduction: Labial herpes simplex lesions (‘‘cold sores’’) are very common mucocutaneous lesions worldwide. Despite the high effectiveness of systemic acyclovir and other nucleoside derived drugs in the management of herpes simplex, topical acyclovir ointments have little impact on the clinical management of the disease. Current topical ointments are not very effective in the control of bacterial coinfection that is commonly observed in clinical practice and increases the amount of pain, edema, erythema, and risk of inesthetic scars and the duration of the crisis. New topical treatments with both an antibacterial and antiherpetic effect would be very useful in the treatment of herpes labialis. Objectives: Evaluate the effects of topical application of silver sulphadiazine 1% 1 cerium nitrate 0.4% (ss 1% 1 cn 0.4%) in the treatment of herpes simplex lesions and compare with topical use of acyclovir ointment. Methods: A prospective, comparative, double-blinded clinical trial was performed in 25 patients with herpes labialis. Thirteen were treated with the SS 1% 1 CN 0.4% compound and the other patients were treated with topical acyclovir ointment for 5 days. The clinical evolution had been compared by Friedman’s and Wilcoxon’s test. Results: Most indices analyzed demonstrated superiority in the application of SS 1% 1 CN 0.4% when compared with topical acyclovir. SS 1% 1 CN 0.4% association was very well tolerated and no side effects either local or systemically were detected. Both edema and erythema were statistically less observed (P \ .034) among the patients treated with SS 1% 1 CN 0.4%; paresthesia was also reduced (P \.08) in the same group after day 5.
Commercial support: None identified.
The realization of this study was sponsored by Silvestre Labs.
AB12
J AM ACAD DERMATOL
Conclusions: The association of SS 1% 1 CN 0.4% proved to be well tolerated in our study. The decrease of erythema, edema and paresthesia in the group treated with SS 1% 1 CN 0.4% was statistically significant after 5 days of the beginning of the treatment when compared with topical acyclovir. We believe that the association of SS 1% 1 CN 0.4% is a promising topical therapy for herpes labialis.
FEBRUARY 2007
P47
Cidofovir diphosphate inhibits molluscum contagiosum DNA polymerase activity Takahiro Watanabe, MD, Department of Dermatology, University of Tokyo, Tokyo, Japan; Tamaki Kunihiko, MD, Department of Dermatology, University of Tokyo, Tokyo, Japan
A double-blind, randomized controlled trial of duct tape for the treatment of common warts in an adult population Rachel Wenner, MD, VA Medical Center, Minneapolis, MN, United States; Sharone Askari, MD, VA Medical Center, Minneapolis, MN, United States; Peter Cham, MD, VA Medical Center, Minneapolis, MN, United States; Erin Warshaw, MD, MS, VA Medical Center, Minneapolis, MN, United States Objective: To evaluate the efficacy of duct tape occlusion therapy for the treatment of common warts.
Molluscum contagiosum (MC) produces a benign papular eruption of multiple umbilicated cutaneous tumors. In HIV-infected individuals, extensive and recalcitrant MC lesions occur that represents a therapeutic challenge. Several studies describe the successful use of cidofovir, a nucleoside analog of deoxycytidine monophosphate, applied topically or by intravenous infusion for otherwise recalcitrant MC. The purpose of this study was to examine the effect of nucleoside analogs on the enzymatic activity. Because molluscum contagiosum virus (MCV) cannot be grown in tissue culture cells and does not infect animals, we have expressed MCV DNA polymerase gene by in vitro transcription-translation. DNA polymerase activity was measured by the incorporation of radioactive deoxycytidine into activated DNA. Twenty to fifty micromolars of CDV diphosphate, an active intracellular metabolite of CDV, was found to inhibit in vitro translated MCV polymerase activity by 50%. CDV diphosphate elicited a similar inhibitory effect on DNA polymerase of herpes simplex virus type 1 and cowpox virus. We believe these results provide for the first time biochemical support for a therapeutic use of cidofovir in the treatment of MC. Commercial support: None identified.
Design: A double-blinded, controlled, clinical intervention trial. Setting: Veterans Affairs Medical Center. Participants: A total of 90 immunocompetent adult volunteers with at least one wart measuring between 2 mm and 15 mm were enrolled in this study between from October 2004 until July 2005; 78 patients completed the study. Intervention: Patients were randomized using a computer-generated code to receive either pads consisting of moleskin with transparent duct tape (active) or moleskin alone (control). Patients were instructed to wear the pads for 7 consecutive days and leave the pad off on the seventh evening. This process was repeated for 2 months or until the wart resolved, whichever occurred first. Follow-up visits occurred at 1 and 2 months. Main Outcome Measure: One hundred percent resolution of the wart. Secondary outcomes included change in size of the target wart and recurrence rates at 6 months for warts with complete resolution. Results: There were no statistically significant differences in the proportions of patients with resolution of the target wart (duct tape 5 8/40 (20%) vs. control 5 9/41 (22%)). Of patients with complete resolution, 75% (6/8) in the duct tape group and 33% (3/9) of those in the control group had recurrence of the target wart by sixth months. Conclusions: Occlusive therapy with transparent duct tape is not significantly better than moleskin alone for treatment of common warts in adults. Limitations: Limitations include the use of an acrylic-based adhesive transparent duct tape. Investigator-initiated study was supported by a Minnesota Medical Foundation Medical Student Grant (non-profit foundation).
P46
P48
Isolation and characterization of the antifungal resistance genes of trichophyton mentagrophytes Aditya Gupta, MD, PhD, Mediprobe Research, Inc, London, Ontario, Canada; Jagpal Singh, PhD, Mediprobe Research, Inc, London, Ontario, Canada; Muhammad Zaman, PhD, Mediprobe Research, Inc, London, Ontario, Canada Trichophyton rubrum and T mentagrophytes are the major fungal pathogens of onychomycosis, a major dermatophytic skin disorder present in 6% to 13% of the North American population resulting in nail plate thickening and discoloration. Previous observations in the literature suggest that T rubrum can acquire increased antifungal resistance, while direct evidence of T rubrum resistance to tioconazole as well as cross-resistance to acridine and ethidium bromide upon increasing exposure to the azole derivative have been reported in vitro. There are also reports that T rubrum can acquire resistance to terbinafine and other squalene epoxidase inhibitors in vitro. The observation that newer and more potent antifungal drugs, such as terbinafine, exhibit a mycological cure rate of 60% to 80% in patients along relapse rates approaching 10% or higher, indirectly suggest that T rubrum drug resistant strains may play an important role in these phenomena. Despite these reports, the molecular mechanisms of T rubrum and T mentagrophytes drug resistance are poorly understood, and a clear understanding of this process is essential for designing new antifungal drugs and for formulating new strategies for effective therapy. Membrane transporters with ATP-binding cassette motifs (ABC transporters) are known to be responsible for conferring multiple drug resistance phenotypes in a wide range of organisms. Our research focuses on the isolation and characterization of ABC transporter genes in T rubrum and T mentagrophytes and investigating their possible involvement in conferring antifungal resistance. First, trichophyton cell lines with reduced fluconazole susceptibility were selected by successive passages in media supplemented with increasing fluconazole concentrations. PolyA1 RNA extracted from these cell lines were then used to clone a cDNA library. Screening of this library using primers specific for human, fungal and bacterial ABC transporter motifs resulted isolation of a single 4kB candidate mdr gene from T mentagrophytes, named Tmmdr1. We are currently completing sequencing of Tmmdr1 to fully confirm its homology to other mdr genes. Functional characterization of Tmmdr1 will be undertaken by transforming and expressing the gene in drug-sensitive Saccaromyces cerevisae strains to determine if it can reverse drug sensitivity. The use of this yeast as an assay organism to select drug-resistant genes is well documented from a wide variety of prokaryotic and eukaryotic species.
Comparative trial of topical application of silver sulphadiazine 1% 1 cerium nitrate 0.4% and acyclovir in labial herpes simplex lesions Omar Lupi, MD, MMSc, PhD, Hospital Universitario Clementino Fraga Filho/UFRJ, Rio de Janeiro/RJ, Brazil; Mariana Gusm~ao, MD, Universidade Federal do Rio de Janeiro, Rio de Janeiro/RJ, Brazil; Ange´lica Gonc¸alves, MD, Universidade Federal do Rio de Janeiro, Rio de Janeiro/RJ, Brazil; Paula Dadalti, MD, MMSc, PhD, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil Introduction: Labial herpes simplex lesions (‘‘cold sores’’) are very common mucocutaneous lesions worldwide. Despite the high effectiveness of systemic acyclovir and other nucleoside derived drugs in the management of herpes simplex, topical acyclovir ointments have little impact on the clinical management of the disease. Current topical ointments are not very effective in the control of bacterial coinfection that is commonly observed in clinical practice and increases the amount of pain, edema, erythema, and risk of inesthetic scars and the duration of the crisis. New topical treatments with both an antibacterial and antiherpetic effect would be very useful in the treatment of herpes labialis. Objectives: Evaluate the effects of topical application of silver sulphadiazine 1% 1 cerium nitrate 0.4% (ss 1% 1 cn 0.4%) in the treatment of herpes simplex lesions and compare with topical use of acyclovir ointment. Methods: A prospective, comparative, double-blinded clinical trial was performed in 25 patients with herpes labialis. Thirteen were treated with the SS 1% 1 CN 0.4% compound and the other patients were treated with topical acyclovir ointment for 5 days. The clinical evolution had been compared by Friedman’s and Wilcoxon’s test. Results: Most indices analyzed demonstrated superiority in the application of SS 1% 1 CN 0.4% when compared with topical acyclovir. SS 1% 1 CN 0.4% association was very well tolerated and no side effects either local or systemically were detected. Both edema and erythema were statistically less observed (P \ .034) among the patients treated with SS 1% 1 CN 0.4%; paresthesia was also reduced (P \.08) in the same group after day 5.
Commercial support: None identified.
The realization of this study was sponsored by Silvestre Labs.
AB12
J AM ACAD DERMATOL
Conclusions: The association of SS 1% 1 CN 0.4% proved to be well tolerated in our study. The decrease of erythema, edema and paresthesia in the group treated with SS 1% 1 CN 0.4% was statistically significant after 5 days of the beginning of the treatment when compared with topical acyclovir. We believe that the association of SS 1% 1 CN 0.4% is a promising topical therapy for herpes labialis.
FEBRUARY 2007