Non-Dermatophyte Molds are not pure Contaminants in Onychomycosis Ranthilaka R. Ranawaka Consultant Dermatologist General Hospital Kalutara, Sri Lanka

Background Onychomycosis is caused by dermatophytes, non- dermatophyte molds (NDM) and Candida spp. NDM are saprophytic molds, which are widely distributed as soil and plant pathogens. In routine practice -the prevalence of NDM onychomycosis was much higher in our setting than reported in the literature. High prevalence of NDM onychomycosis has been reported from India (22%), Malaysia (35.5%), Thailand (51.6%) and Pakistan (68%), too.

Two studies were conducted in Sri Lanka, 2004 (128 patients) and 2012 (178 patients) In both studies mycology was performed at Department of Microbiology, Faculty of Medicine Karapitiya, Galle.

What ever the causative fungus onychomycosis was clinically indistinguishable 30, man, chemist, gardening, Aspergillus flavus isolated 36,housewife, with dystrophic big toe nails for two years. Fusarium dimerum isolated. 55, labourer woman, 10 years, Penicillium spp isolated

Dermatophytic and non-dermatophytic onychomycosis was clinically indistinguishable in our case series. 56, farmer, Trichophyton metagrophytes isolated 67, male, gardening, Epidermophyton spp isolated 54, mid wife, Epidermophyton spp isolated

Concomitant paronychia in 2004 2012 NDM 61% 66.6% Candida 38% 56.6% 52, manual labourer, Fusarium oxysporum isolated. 34, man, mechanic, Aspergillus flavus isolated

Diagnosis Microscopy – High false negative results Culture -'gold standard' of mycological diagnostics. approximately 30% false negative results Histology- PAS stain Periodic acid-Schiff (PAS) stained nail clippings - second line diagnostic tool. Information concerning the vitality of the fungi and accurate identification of the specific pathogen is not available through this investigation alone.

Sample collection Correct sample collection is important to get high microscopy positive results The positivity rate for the shaving method was 96.8%, while the rate for the conventional method (nail clipping) was 48.4%. Reference- HKTA Gunasekera, A Nagahawatta, N de Silva. An alternative technique for collecting nail specimens for mycological investigations. Sri Lankan Journal of Infectious Diseases 2011 Vol.1 (1); 24-26 DOI: http://dx.doi.org/10.4038/sljid.v1i1.3186

Treatments Itraconazole pulse therapy Terbinafine pulse therapy Itraconazole 200mg b.i.d for 7 days per month Terbinafine pulse therapy Terbinafine 250mg b.i.d for 7 days per month 2 pulses for finger nails and 3 pulses for toe nails Topical antifungal (3% thymol or clotrimazole lotion) continued twice a day application for total of 12 months

Primary end point At the end of 2 or 3 pulses Secondary end point At the end of 12 months follow-up period

Table. Isolated fungi in 128 patients with onychomycosis (2004) Species Total fungal isolates Both microscopy and culture positive Microscopy negative, culture positive Dermatophytes Trichophyton rubrum T. mentagophytes 17 11 6 17 (20) 11 (12.9) 6 (7) - Yeasts Candida albicans Other Candida sp Other yeasts 31 4 20 7 29(34.1) 2 (2.35) 20(23.5) 7 (8.2)   2 Non- Dermatophyte Molds (NDM) Aspergillus niger A.flavus A.terreus Penicillium sp Fusarium sp Cladosporium sp Rhizopus sp Acremonium sp Paecilomycis sp Cylindrocarpon sp 68 47 12 8 1 39(45.8) 19(22) 5(6) 2(2.35) 3(3.5) 3 29 18 No growth Mixed infections 8 (6%) Total 128 85(66.4) 31(24.2%) Species Total fungal isolates Both microscopy and culture positive Microscopy negative, culture positive Dermatophytes Trichophyton spp Mentagrophytes spp Epidermophyton spp 15 8 5 2 12 (17.6) 6 4 1 - Candida species Candida albicans Non-albicans 32 13 19 21 (30.9) 11 Non-dermatophyte molds Aspergillus niger flavus Aspergillus spp Penicillium spp Fusarium spp Scopulariopsis brevicaulis Cladosporium spp Paecilomyces spp 101 44 9 3 35 (51.5) 10 (14.7) 3 (4.4) 8 (11.7) 1 (1.5) 2 (2.9) 66 34 22 Bacterial growth No growth Missing reports 7 18 Total 178 68 (45.9) 80 (54.0)

Results Fusarium onychomycosis Itraconazole Terbinafine Total number of patients treated (n=9) 6 3 Total number of nails treated (n=45) 25 20 Cured at the end of Rx (number of nails) none Clinical cure at 12 months 52% 50% Mycological cure 66.6% Recurrence in 3 months 30.7% (4/13) Recurrence in 12 months 61.5% (8/13) Ranawaka RR, de Silava N, Ragunathan RW. Onychomycosis caused by Fusarium sp p in Sri Lanka: prevalence, clinical features and response to itraconazole pulse therapy in six cases. J Dermatolog Treat.2008;19(5):308-12 Ranawaka RR, Nagahawatte A, Gunasekara TA. Fusarium Onychomycosis: prevalence, clinical presentations, response to itraconazole and terbinafine pulse therapy and one-year follow-up in 9 cases. Accepted for publication in International Journal of Dermatology

Treated with two pulses of terbinafine. Complete cure after 12 months Recurrence of nail destruction after 19 months Before treatments 68, woman, only left thumb nail, for 4 yrs, Fusarium dimerum isolated. Terbinafine additional pulse given. Continued topical 3% thymol Normal nail growing underneath- 21 months

Before treatments 43-year-old pre-school teacher with nail dystrophy on 4th & 5th finger nails on L/hand and 1st, 4th & 5th nails on R/hand. Fusarium oxysporum was isolated. After 12 months After 12 months of follow-up, 3 nails on R/ hand cured. Additional pulse of terbinafine given.

Results- cont.. NDM onychomycosis Itraconazole Terbinafine Total number of patients completed the study (n=57) 30 27 Total number of nails treated (n=249) 152 97 Cured at the end of Rx (number of nails) (n=16 nails) 14 (9.2%) 2 (2.0%) Clinical cure at 12 months (n=148 nails) 95 (62.5%) 53 (54.6%) Recurrence within 12 months (n=14) 10 (6.57%) 4 (4.1%) Side effects Exacerbated gastritis n=2 Stopped Rx Nausea on two, but continued Rx Ranthilaka RR, de Silva N, Ragunathan RW. Non-dermatophyte mold onychomycosis in Sri Lanka. Dermatology Online Journal 2010; 18 (1): 7 Ranawaka RR, Nagahawatte A, Gunasekara TA, Weerakoon HS. Randomized, double blind, comparative study on efficacy and safety of itraconazole pulse therapy and terbinafine pulse therapy on Non-Dermatophyte Mold onychomycosis (on publication process)

Treatment response in different NDM species Aspergillus spp (n=46) Fusarium spp (n=8) Penicillium spp (n=2) Scopulariopsis spp Treatments I T Total number of patients completed the study 21 25 6 2 - 1 Total number of nails completed Rx 107 89 24 8 13 Clinical cure rate (number of nails) 73/107 (68.22%) 49/89 (55.05%) 12/24 (50%) 4/8 11/13 (84.6%) No cure Recurrence within 12 months 6/107 (5.6%) 5/89 2/24 (8.3%) 1/8 (12.5%) I- Itraconazole pulse therapy, T- Terbinafine pulse therapy

Cured at 12 months 54, house wife, gardening, Aspergillus flavus isolated. Treated with 3 pulses of Terbinafine. . Toe nails were not cured

32, House wife, 3 years, Aspergillus niger isolated. Treated with itraconazole. 70% clinical improvement at12 months One finger nail was also affected which did not improve with treatments

62-year old retired clerk, who engaged in gardening on bare hand, had this nail destruction for >6 years. Aspergillus niger was isolated. She was treated with three pulses of Itraconazole. After 12 months of follow-up her finger nails were completely cured. She also had Candida infection in finger webs.

73, house wife, R/middle finger dystrophy for one year, A 73, house wife, R/middle finger dystrophy for one year, A. niger isolated. Rx- Itraconazole 3 pulses R/ middle finger cured after 12 months She also had B/L big toe nails involved, which were not cured.

Summary NDM and Candida are the prevalent fungi in onychomycosis in our setting These fungi earlier thought to be related to immune suppression or diabetes is disputed Nail shavings gives higher microscopic positivity than nail clippings NDM showed almost equal efficacy to both Itraconazole and Terbinafine pulses (I>T) Both treatments showed only 50-60% cure rate Recurrence was common with itraconazole

Summary – cont…. Additional pulse recommended depending on the response Aspergillus spp and Penicillium spp showed better response to Itraconazole Therefore species identification prior to therapy may help to select the effective drug Both Itraconazole (SLR 1500- 3500 per week) and Terbinafine (SLR 1300 per week) are expensive therefore before starting treatments you may educate patients on efficacy of current antifungal available Future research should focus on finding more effective antifungal for the treatment of non dermatophyte onychomycosis.

J Am Acad Dermatol. 2000 Feb;42:217-24 Fusarium spp 26 combination 40% Authors Journal Species patients Rx Clinical cure Gianni C1 Dermatology. 2004;209(2):104-10 Aspergillus spp 34 Terbinafine 88% Tosti A J Am Acad Dermatol. 2000 Feb;42:217-24 Fusarium spp 26 combination 40% 7 100% S brevicaulis 17 69.2% Br J Dermatol. 1996 Nov;135(5):799-802 Scopulariopsis 6 3-itraconazole 3- terbinafine One patient Gupta AK Dermatology. 2001;202(3):235-8 59 itraconazole terbinafine 12/12 J Cutan Med Surg. 2001 May-Jun;5(3):206-10 5 2/4 0/1 2 1/1 3/6 Cohen AD J Dermatolog Treat. 2003 Dec;14(4):237-42 toenail onychomycosis (dermatophytes) 117 43-itraconazole 74-Terbinafine No difference in MC or CC Combination- combining different treatments (systemic itraconazole, systemic terbinafine, topical terbinafine after nail plate avulsion, and ciclopirox nail lacquer)

Authors Journal Species Shemer A Dermatol Ther. 2012 Nov-Dec;25(6):582-93 Dermatophytes Terbinafine is superior to itraconazole for dermatophyte onychomycosis Gupta AK Am Acad Dermatol. 2000 Oct;43(4 Suppl):S70-80 Ciclopirox nail lacquer for the treatment of finger and toe onychomycosis Dermatology. 2001;202(3):235-8 NDM Griseofulvin is ineffective against toe onychomycosis caused by S. brevicaulis Ketoconazole is not recommended for toe onychomycosis given its potential for adverse effects Yin Z J Dermatolog Treat. 2012 Dec;23(6):449-52 toenail onychomycosis Itraconazole therapy is more likely to produce mycological recurrence compared with terbinafine therapy. J Cutan Med Surg. 2013 May-Jun;17(3):201-6 Dermatophytes- followed-up for 1.25 to 7 years Itraconazole therapy was associated with higher RRs than terbinafine therapy Additional pulses administered depending upon the response

Thank you Dr Ajith Nagahawatte (co-investigator) Dr Nelum de Silva (co-investigator) Mr. Aravinda Gunasekara (co-investigator) Department of Microbiology, Faculty of Medicine, Karapitiya, Galle.