Randomized, double-blind, comparative study on ...

Journal of Dermatological Treatment

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Randomized, double-blind, comparative study on efficacy and safety of itraconazole pulse therapy and terbinafine pulse therapy on nondermatophyte mold onychomycosis: A study with 90 patients Ranthilaka R. Ranawaka, Ajith Nagahawatte, Thusitha Aravinda Gunasekara, Hema S. Weerakoon & S. H. Padmal de Silva To cite this article: Ranthilaka R. Ranawaka, Ajith Nagahawatte, Thusitha Aravinda Gunasekara, Hema S. Weerakoon & S. H. Padmal de Silva (2015): Randomized, double-blind, comparative study on efficacy and safety of itraconazole pulse therapy and terbinafine pulse therapy on nondermatophyte mold onychomycosis: A study with 90 patients, Journal of Dermatological Treatment To link to this article: http://dx.doi.org/10.3109/09546634.2015.1119781

Published online: 10 Dec 2015.

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Date: 11 December 2015, At: 08:08

http://informahealthcare.com/jdt ISSN: 0954-6634 (print), 1471-1753 (electronic) J Dermatolog Treat, Early Online: 1–9 ! 2015 Taylor & Francis. DOI: 10.3109/09546634.2015.1119781

ORIGINAL ARTICLE

Randomized, double-blind, comparative study on efficacy and safety of itraconazole pulse therapy and terbinafine pulse therapy on nondermatophyte mold onychomycosis: A study with 90 patients Ranthilaka R. Ranawaka1, Ajith Nagahawatte2, Thusitha Aravinda Gunasekara2, Hema S. Weerakoon1, and S. H. Padmal de Silva3 1

Department of Dermatology, Base Hospital Homagama, Sri Lanka, 2Department of Microbiology, University of Ruhuna, Galle, Sri Lanka, and Department of Evaluation and Research, National Institute of Health Sciences, Kalutara, Sri Lanka

Downloaded by [Ranthilaka Ranawaka] at 08:08 11 December 2015

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Abstract

Keywords

Background: Nondermatophyte mold (NDM) onychomycosis shows poor response to current topical, oral or device-related antifungal therapies. This study was aimed to determine the efficacy and safety of itraconazole and terbinafine pulse therapy on NDM onychomycosis. Methods: Mycologically proven subjects were treated with itraconazole 400 mg daily or terbinafine 500 mg daily for 7 days/month; two pulses for fingernails and three pulses for toenails(SLCTR/2013/013). Results: One-hundred seventy-eight patients underwent mycological studies and 148 had positive fungal isolates. NDM were the prevailing fungi, 68.2%, followed by candida species 21.6%, and dermatophytes made up only 10.1%. Out of NDM Aspergillus spp (75.1%) predominated followed by 8.9% Fusarium spp and 4.95% Penicillium spp. The clinical cure at completion of pulse therapy was statistically significant 9.2% versus 2.0% (p50.05) in itraconazole group. But no statistically significant difference was detected between the two regimens at the end of 12 months; 65.1% versus 54.64%. Recurrences observed in both groups (6.5% vs. 4.1%) were not statistically significant. With itraconazole pulse 68.22% Aspergillus spp, 50.0% Fusarium spp and 84.6% Penicillium spp showed clinical cure, while terbinafine pulse cured 55.0% Aspergillus spp and 50.0% Fusarium spp. Conclusions: NDM was the prevailing fungi in onychomycosis in Sri Lanka. Both itraconazole and terbinafine were partially effective on NDM onychomycosis showing a clinical cure of 54–65%. Future research should focus on searching more effective antifungal for NDM onychomycosis.

Nondermatophyte mold, onychomycosis, itraconazole, terbinafine, pulse therapy, Sri Lanka

Background Onychomycosis is caused by dermatophytes, nondermatophyte molds (NDM) and candida species. The incidence of onychomycosis due to nondermatophyte molds (NDM) is increasing (1–4). NDM shows poor response to current topical, oral or devicerelated antifungal therapies (5,6). Gupta et al. (5) conducting an open-labeled clinical trial demonstrated that itraconazole showing the better efficacy against onychomycosis of the toenails caused by Scopulariopsis brevicaulis (n ¼ 4) and Aspergillus species (n ¼ 6). In the study conducted by Ranawaka et al. (7), with oral itraconazole pulse therapy Fusarium spp (n ¼ 5) showed 60% clinical cure at 12 months follow-up, and the mycological cure was 100%. In onychomycosis caused by Aspergillus spp 12 months after the start of therapy with pulsed terbinafine, clinical

Correspondence: Ranthilaka R. Ranawaka, 310/4, Kulasiri Kumarage Mawatha, Katuwana, Homagama, Sri Lanka. Tel: 0094 718186148. Fax: 0094 112910481. E-mail: [email protected]

History Received 11 June 2015 Revised 31 July 2015 Accepted 24 October 2015 Published online 9 December 2015

and mycological recovery was confirmed in 30 of the 34 patients (88%) (8). Terbinafine is believed to have good in vitro antifungal activity against Scopulariopsis species (9). In a series of eight patients with NDM onychomycosis who were resistant to multiple conventional topical and systemic treatments topical amphotericin B was an efficacious, safe, cheap and easy-to-apply treatment showing clinical cure in all after 12 months treatments; mycological cure was obtained in all but one patient (10). Multicenter clinical trial conducted in US and non-US countries (2000) demonstrated that ciclopirox nail lacquer 8% topical solution was significantly more effective than placebo in the treatment of finger and toe onychomycosis caused by dermatophytes and candida spp; once-daily application for 48 weeks showed mycologic cure rate in 36% versus 9% in the ciclopirox and vehicle groups, respectively. In the same clinical trial, ciclopirox nail lacquer demonstrated a broad spectrum of activity with efficacy against candida species and some nondermatophytes in non-US studies (11). This randomized, double-blind, comparative clinical trial was aimed to determine the efficacy and safety of itraconazole pulse therapy and terbinafine pulse therapy on nondermatophyte mold onychomycosis.

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R. R. Ranawaka et al.

J Dermatolog Treat, Early Online: 1–9

Patients and methods The study was approved by the Ethics Review Committee, Sri Lanka Medical Association, Colombo, Sri Lanka. This clinical trial is registered at Sri Lanka Clinical Trial Registry (SLCTR/2013/013).


Study population Individuals were recruited from those attending the dermatology clinic at the General Hospital Chillaw, Sri Lanka and Base Hospital Homagama, Sri Lanka over a period of 24 months from May 2012 to April 2014. Mycological tests were conducted at the Department of Microbiology, Faculty of Medicine, Karapitiya, Galle, Sri Lanka (Figure 1). The patients who had clinically suggestive onychomycosis were invited to participate. Pregnant women, breast-feeding mothers, patients with known renal impairment, known liver impairment and/or congestive cardiac failure were excluded. Informed written consent was obtained from all who agreed to participate. Patients’ demographic data, occupation, leisure activities (e.g. gardening in bare hand), predisposing factors and concurrent Figure 1. Flow diagram of the participants.

medical and skin diseases were recorded. Frequently worn shoes (open slippers, closed shoes or barefoot), frequent contact with water, soil, mud or chemicals were recorded. Type of onychomycosis was recorded depending on the clinical appearance at presentation: distal and lateral subungual onychomycosis (DLSO), proximal subungual onychomycosis, superficial white onychomycosis and total dystrophic onychomycosis. Liver transaminases (serum glutamic oxaloacetic transaminase (SGOT)/serum glutamic pyruvic transaminase (SGPT)) were performed before and at the end of the therapy, other investigations were performed only if clinically demanded. Photographs of the affected nails were taken before and after treatments. Randomization, blinding The patients’ whom mycology had proven nondermatophyte mold onychomycosis were included in the clinical trial. The patients who consented to participate in the study and who fulfilled the inclusion and exclusion criteria were assigned to the two treatment groups. The treatment options were documented separately and packed in covered opaque envelopes consecutively numbered according to the randomization schedule as to have a

194 paents were invited to parcipate

184 consented to parcipate Excluded following applicaon of exclusion criteria (n=6) 178 underwent mycological studies

Total fungal isolates (n=148)

ExcludedBacterial growth (n=7) No growth (n=18) Mixed growth (n=5)

Total Non-Dermatophyte Mold isolates (n=101) Both microscopy and culture posive (n= 35) Microscopy negave culture posive (n=66) ExcludedRefused oral drugs (n=10) Became pregnant (n=1) Randomized (n=90) in to two treatment groups (1: 1 rao)

Group 1 Itraconazole pulse therapy Number of paents (n=43) Number of affected nails (n=222) Defaulted treatments (n=7) Lost to follow-up (n=4) Stopped treatments due to side effects (n=2) Completed the study (n=30)

Group 2 Terbinafine pulse therapy Number of paents (n=47) Number of affected nails (n=180) Defaulted treatments (n=14) Lost to follow-up (n=6) Completed the study (n=27)

57 paents with NDM completed the study

Group 1 Itraconazole pulse therapy Number of paents (n=30) Number of affected nails (n=169)

Group 2 Terbinafine pulse therapy Number of paents (n=27) Number of affected nails (n=97)

NDM onychomycosis treatments

DOI: 10.3109/09546634.2015.1119781

ratio of 1:1 for itraconazole and terbinafine treatment groups. The allocation sequence was concealed from the researcher enrolling and assessing the participants in sequentially numbered, opaque and sealed and stapled envelopes that were impermeable even to intense light. The participants and the investigator (outcome assessor) were blind to the type of therapy. Same investigator performed clinical assessment on all the participants at each visit until cure.


Sampling method and sample size This study comes under the category of equivalence studies, where there is no major difference between the effectiveness of the treatments but one has practical advantages such as less cost. Therefore, the sample size required was large. Approximately, 473 nail samples per group were needed to detect a treatment difference of 5% with lowest cure rate of one group being 90%. Comparative clinical trial on itraconazole and terbinafine has not been conducted before in Sri Lanka. Both drugs were expensive cost SLRs 1050 (USD 9) for terbinafine and SLRs 1680–7000 (USD 14–50) for itraconazole per seven days course per one patient. Limitations Sample size of this study was limited by the cost of the drugs, cost for fungal cultures, and the available patients within 24 months of the study period. Mycology Nail shavings and subungual debris were collected from the diseased nail. In patients with DLSO, the specimens were obtained from the most proximal portion of the affected nail. Specimens of nails were clarified in 20–30% potassium hydroxide for 10–30 minutes depending on the thickness of the nail specimens. Direct microscopy was done to detect fungal hyphae, spores and yeasts. Each specimen was inoculated on to two Sabouraud’s dextrose agar plates, one with cyclohexamide and the other without. Cultures were incubated at 26 C for 2–3 weeks and were examined twice a week for growth. The relevance and significance of the culture isolates were determined in correlation with the direct microscopic findings of the nail specimens. Tease mount preparation of the colonies in lactophenol cotton blue were used for generic identification of the fungal isolates. Further testing with slide cultures was done for species identification. All NDM samples were done in duplicate.

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Diagnosis of NDM onychomycosis was made based on the following criteria: (1) Nail abnormalities consistent with the diagnosis (2) Positive potassium hydroxide preparations with the presence of hyphae in the nail keratin (3) Growth of the same mold in duplicate cultures (4) Failure to isolate dermatophytes or yeasts in culture (5) Colony morphology and sporulation pattern. Significant growth was considered when at least four of the five criteria were positive, including potassium hydroxide preparation for direct microscopy and isolation of the organism in culture. Treatments The patients were treated with either 400 mg itraconazole or 500 mg terbinafine in divided doses for 7 days per month (1 week on and 3 weeks off monthly pulses). Two pulses were prescribed for fingernails and three pulses for toenails. Trimming the diseased nail short in the case of DLSO was advised, but mechanical debridement was not allowed, as it would further push fungal debris proximally. Both groups were treated with topical 3% thymol in spirit or clotrimazole lotions depending on availability. Patients were followed up in the skin clinic monthly until completion of treatment, then every 3 months for a total of 12 months (Figures 2–6). Efficacy of therapy Efficacy of therapy was judged by both clinical and mycological cure. Clinical cure was defined as complete absence of all the clinical signs of onychomycosis: onycholysis, subungual hyperkeratosis, pain or paronychia. As black or brown discoloration persisted even after normal nail growth for a few more months, we disregarded it as a sign of clinical cure. Mycological cure was defined as negative direct microscopy and culture. Statistical analysis The analysis was performed using descriptive statistical methods. Rates and percentages were calculated where relevant and the observed differences within these two groups were assessed for any statistically significant differences using Chi square test. The observed outcome was assessed using a percentage and statistical significance was assessed using Chi square test and Z scores for comparison of rates. Statistical software package SPSS version 17 was used for the analysis.

Figure 2. Sixty-eight-year-old housewife, with dystrophic left thumb nail only for more than 4 years. Fusarium dimerum was isolated (A). She is treated with two pulses of terbinafine. After 12 months of follow-up, her fingernail is completely cured (B). But nail destruction recurred 9 months after confirming cure (C).

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Figure 3. Fifty-four-year-old housewife with dystrophic bilateral big toe nails for 6 years and L/4th finger for 6 months (A). Aspergillus flavus was isolated. She was treated with three pulses of Terbinafine. After 12 months of follow-up her fingernail cured completely (B) while toe nails remained same (C).

Figure 4. Thirty-two-year-old house wife with dystrophic bilateral big toe nails for 3 years (A) and R/4th finger for 8 months (B). Aspergillus niger was isolated. She was treated with three pulses of Itraconazole. After 12 months of follow-up, her toe nails showed 70% clinical improvement (C) while finger nail remained same.

Figure 5. Sixty-two-year-old retired clerk, who engaged in gardening on bare hand, had this nail destruction for more than 6 years (A). Aspergillus niger was isolated. She was treated with three pulses of Itraconazole. After 12 months of follow-up, her fingernails and toe nails were completely cured (B).

Figure 6. Seventy-three-year-old housewife with R/middle finger dystrophy for 1 year (A) and bilateral big toe nails dystrophy for 5 years. Aspergillus niger was isolated. She was treated with three pulses of Itraconazole. After 12 months of follow-up, her fingernail is completely cured (B), while toe nails remained same (C).


DOI: 10.3109/09546634.2015.1119781

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Table 1. Isolated fungi in 178 patients with onychomycosis.


Species Dermatophytes Trichophyton spp Mentagrophytes spp Epidermophyton spp Candida species Candida albicans Nonalbicans Nondermatophyte molds Aspergillus niger flavus Aspergillus spp Penicillium spp Fusarium spp Scopulariopsis brevicaulis Cladosporium spp Paecilomyces spp Bacterial growth No growth Mixed growth Total

Total fungal isolates

Both microscopy and culture positive

Microscopy negative, culture positive

15 8 5 2 32 13 19 101 44 (43.5) 32 (31.6) 4 5 9 2 3 2

12 (17.6) 6 4 2 21 (30.9) 13 8 35 (51.5) 10 (14.7) 10 (14.7) 3 (4.4) 8 (11.7) 1 (1.5) 1 (1.5) 2 (2.9)

2 1 -

66 34 22 4 2 1 1 2 -

148

68 (45.9)

80 (54.0)

11

7 18 5

Bold values indicate total values in dermatophytes, candida species and nondermatophyte molds.

Results Out of 178 patients who underwent mycological studies, 148 had positive fungal isolates. Nondermatophyte molds were the mostly isolated fungi, 68.2% (n ¼ 101), followed by candida species 21.6% (n ¼ 32) and dermatophytes made up only 10.1% (n ¼ 15) (Table 1). Out of NDM, Aspergillus spp (75.1%) predominated followed by 8.9% Fusarium spp and 4.95% Penicillium spp.

Eczema (20.0%, n ¼ 18) was the most common coexisting skin disease, while candida toe webs was seen in three. Twenty-seven (30.0%) patients had medical problems, where thyroid disease in fifteen, varicose veins in four, and diabetes in one. All were otherwise healthy and immune-competent. Mold onychomycosis was not significantly associated with systemic diseases or immune suppression in our study population.

Study sample

Treatment response

Only NDM isolates were included in the present clinical trial (flow chart). Although microscopy negative samples were considered contaminants by the definition we used, if it was clinically indicative we included them in this clinical trial. There were five patients with mixed growth, either two NDM spp or NDM with Candida spp, whom were not included in this clinical trial. Ninety patients who had NDM isolation were randomized in 1:1 ratio into two treatment groups (flow chart). Twenty-one defaulted therapy and 10 were lost to follow-up and were excluded from the final analysis. Two patients whom were treated with itraconazole had worsened their gastritis during treatments and the therapy was withheld. Finally, total of 57 (63.3%) patients with 266 diseased nails completed the clinical trial.

The clinical cure (CC) at completion of pulse therapy was statistically significant 9.2% versus 2.0% (p50.05) in itraconazole group compared to terbinafine group. But no statistically significant difference was detected between the two regimens at the end of 12 months; itraconazole pulses (65.1% CC) and terbinafine pulses (54.64% CC). Recurrences observed in itraconazole group were higher (6.5%) than in terbinafine group (4.1%), although the difference was not statistically significant (p40.05) (Table 3). Repeat mycology was performed at 12 months from one affected nail from each patient and showed mycological cure (MC) in 23 out of 30 (76.66%) patients in itraconazole group and 21 out of 27 (77.77%) in terbinafine group. Clinical cure of different NDM species is shown in Table 4. Although Aspergillus spp showed better CC with itraconazole (68.22% vs. 55.05%), the difference was statistically insignificant (p40.05). Out of eight Fusarium isolates, six were treated with itraconazole and the other two were with terbinafine and showed equal efficacy with both drugs showing 50% CC (p40.9). While the two patients with Penicillium spp showed 84.6% CC with itraconazole pulses, Scopulariopsis spp (n ¼ 1) was not cured at all with itraconazole. We compared treatment response in microscopy positive samples with microscopy negative samples (Table 5) and showed that both samples showing equivalent CC (60% vs. 59.1%). Even though statistically insignificant microscopy negative samples showed higher recurrence rate (6% vs. 4.7%)

Demographic data and clinical manifestations of the study group (n ¼ 90) Women predominated, men:women ratio of 1:2 (Table 2). The mean (median) age of the study participants’ was 47.57 (49) years ranged 20 –80 years. Mean (median) duration of the disease was 38.49 (24) months, ranged 1 month to 20 years. Nearly, one-third of affected were housewives (32.2%), followed by professionals (31.1%). Nearly, half of our study group (42.2%) walked barefoot at least at home garden. Frequent contact with water was the predominantly identified predisposing factor in fingernail disease, while barefoot walking in toe nails. Distal and lateral subungual onychomycosis was the commonest (77.78%) clinical presentation, followed by total onychodystrophy in 16.78%. Paronychia was associated in 55.56% of subjects. Nearly, 80% (77.78%, n ¼ 70) had their big toe nails diseased and thumb was involved in 58.89% (n ¼ 53), both toes and finger nails were involved in 46.67% (n ¼ 42).

Discussion NDM are filamentous fungi that are commonly found in nature as soil saprophytes and plant pathogens. Nail invasion by NDM was

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Table 2. NDM onychomycosis: demographic data and clinical manifestations of the study group (n ¼ 90).

Total number of patients recruited to the study Total number of nails treated Patients defaulted treatments


Stopped treatments due to side effects

Total number (percentage)

Group 1 Itraconazole pulse

Group 2 Terbinafine pulse

90 402 21 (23.33)

43 222 7

47 180 14

2 (2.2)

worsened gastritis in two, stopped therapy

nausea in two patients, but continued therapy

p value and significance

X2 ¼ 1.435 p ¼ 0.23 p40.05 X2 ¼ 2.135 p ¼ 0.144 p40.05 X2 ¼ 0.217 p ¼ 0.64 p40.05 X2 ¼ 0.329 p ¼ 0.566 p40.05

Patients lost to follow-up

10 (11.1)

4

6

Patients completed the study

57 (63.33)

30

27

Sex distribution Men women

30 (33.33) 60 (66.67)

13 30

17 30

X2 ¼ 0.356 p ¼ 0.551 p40.0

18 23 21 16 12

7 12 10 9 5

11 11 11 7 7

X2 ¼ 1.388 p ¼ 0.846 p40.05

29 (32.2) 28 (31.1) 4 (4.4) 5 (5.5) 3 (3.3) 9 (10.0)

14 12 2 3 1 4

15 17 2 2 2 5

X2 ¼ 0.3265 p ¼ 0.85 p40.05

38 (42.2) 22 (24.4) 4 (4.4) 8 (8.89)

18 12 3 3

20 10 1 5

X2 ¼ 0.2871 p ¼ 0.866 p40.05

Age distribution Less than 35 36–45 46–56 57–65 66–80 Occupational distribution Housewives Professionals (clerk, lecturer, teacher, shop keeper, nurses) Manual laborers Housemaid, child caring farmers others (mason, mechanic, carpenter, security) Predisposing factors identified Barefoot walking Gardening Farming Closed shoes Clinical manifestations DLSO TOD PSO Associated paronychia Site of involvement Big toe nails Thumb involved Finger nails only Both toes and finger nails

70 (77.78) 16 (16.78) 3 (3.3) 50 (55.56)

36 5 1 25

34 11 2 25

All four p40.05 NS

70 (77.78) 53 (58.89) 21 (23.3) 42 (46.67)

36 30 6 23

32 23 15 19

X2 ¼ 4.466 p ¼ 0.107 p40.05

Duration of the disease 51 month 1–3 4–6 7–12 13–24 months 42 yrs–4 yrs 5–10 yrs 10–20 yrs

0 8 (8.89) 11 (12.2) 24 (26.67) 16 (17.78) 14 (15.5) 10 (11.1) 6 (6.67)

0 3 5 10 10 5 5 4

0 5 6 14 6 9 5 2

X2 ¼ 3.2626 p ¼ 0.515 p40.05

Associated medical problems Diabetes Varicose veins Thyroid disease Hypertension Atopy, asthma Breast carcinoma 4 years back Hyper IgE syndrome

7 1 4 15 (16.67) 4 1 1

3 1 3 7 1 0 0

4 0 1 8 3 1 1

X2 ¼ 4.612 p ¼ 0.326 p40.05

Associated skin diseases Eczema Candida toe webs Rubber contact dermatitis Cement contact dermatitis

18 (20.0) 3 2 1

11 1 1 0

7 2 1 1

X2 ¼ 2.069 p ¼ 0.558 p40.05


DOI: 10.3109/09546634.2015.1119781

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Table 3. NDM onychomycosis: treatment response to itraconazole pulse therapy and terbinafine pulse therapy. Treatments

Itraconazole group

Total number of patients completed the study Total number of nails completed the study Clinical cure at the end of treatments (number of nails)

Clinical cure at 12 months (number of nails)

Mycological cure at 12 months (number of patients)

30 152 14 (9.2%)

27 97 2 (2.0%)

99 (65.1%)

53 (54.64%)

23/30 (76.66%)

Side effects (number of patients) Worsened gastritis in patients - stopped treatments Nausea – continued treatments Recurrence (number of nails)


Terbinafine group

Statistical significance

Z score ¼ 2.17 p ¼ 0.034 p50.05 Statistically significant Z score ¼ p ¼ 0.30 p40.05 Not statistically significant p40.05 Not statistically significant

21/27 (77.77%)

2 0 10 (6.5%)

0 2 4 (4.1%)

Z score ¼ 0.79 p ¼ 0.44 p40.05 Not statistically significant

Table 4. NDM onychomycosis: treatment response in different NDM species. NDM species Treatments

Aspergillus spp I

Fusarium spp T

I

6 25 21 Total number of patients treated 24 89 107 Total number of nails completed treatments 73/107 (68.22%) 49/89 (55.05%) 12/24 (50%) Clinical cure rate (number of nails)

Recurrences (number of nails)

Penicillium spp

Scopulariopsis spp

T

I

T

I

T

2 8 4/8 (50%)

2 13 11/13 (84.6)

–

1 6 No cure –

–

Z score ¼ 1.16 p ¼ 0.24 (p40.05) 6/107 (5.6%) 5/89 (5.6%)

Z score ¼ 0.00 (p40.9) 2/24 (8.3%) 1/8 (12.5%)

Z score ¼ 0.003 p ¼ 0.99 (p40.05)

Z score ¼ 0.33 p ¼ 0.74 (p40.05)

–

–

I- Itraconazole pulse therapy, T- Terbinafine pulse therapy. Table 5. NDM onychomycosis: comparative treatment response in microscopy positive and microscopy negative samples. Microscopy positive Total number of patients treated Total number of nails treated Clinical cure at 12 months (number of nails)

Recurrence rate (number of nails)

20 85 51/85 (60%)

4 (4.7%)

considered uncommon with prevalence rate ranging from 1.45% to 17.6% (12,13). In our study population, NDM was the highest followed by candida spp, where dermatophytes comprised only 10.1%. These results enhanced author’s findings in 2005 (2), where NDM (45.8%) was the most frequently isolated fungi in onychomycosis, followed by yeasts (34.1%); dermatophyte infection made up only 20% and proved that NDM is no longer pure contaminants. High prevalence of NDM onychomycosis had been reported from India (22%) (14) Malaysia (35.5%) (15), Thailand (51.6%) (16) and Pakistan (68%) (17), too. Although NDM and candida can invade healthy nails, secondary colonization of previously damaged nail is common.

Microscopy negative 37 164 97/164 (59.1%)

10 (6%)

Statistical significance

Z score ¼ 0.08 p ¼ 0.93 p40.05 Not statistically significant Z score ¼ 0.44 p ¼ 0.67 p40.05 Not statistically significant

Barefoot walking which is a common practice in tropical climate results frequent minor damages to nails and exposure to contaminated soil predisposes them to pathogenic saprophytic fungi. Frequent contact with water during household work in housewives was the commonest predisposing factor in women. In our case series, except for two patients who had diabetes, all were immunocompetent. Therefore, immune suppression in NDM was disputed in our setting. Since NDM is considered common contaminants both direct microscopy and culture should be positive to diagnose a significant growth. Direct microscopy of nail specimen is an important step in the laboratory diagnosis of NDM onychomycosis. Sensitivity of this test is aided by the collection of good


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specimen. Collection of samples from dystrophic and hyperkeratotic nail is a tedious task. Conventional methods, that is nail clipping has disadvantages, such as it does not give good specimens from hyperkeratotic and very dystrophic nails those are common in manual laborers and agricultural workers, nail clippers are sometimes not wide enough to encompass the thickness of the nail, clippings take longer time to clarify in KOH and microscopy is less sensitive if clarification does not provide a single layer of cells to observe fungal elements. To overcome these problems, nail shaving method was developed by Gunasekara et al. (18). With shaving method, extremely thin shaving samples could be obtained which were clarified in KOH easily. Sensitivity of the shaving method was determined as 96.8% while sensitivity of the clipping method was 79.5% (18). We used shaving method in our study. In the shaving method, the technique of obtaining thin nail shavings was easy, did not cause any discomfort to the patient. Nail shavings were extremely thin, did not require prolonged clarification and could be examined almost immediately which gave a clear single celled layer of the nail maintaining the architecture of the nail to visualize filaments easily. Even with shaving method, direct microscopy was negative in 54.0% of our sample. Since microscopy and culture are performed on two different nail specimens, unlike in other pathological specimens, such as urine, sputum, stool or cerebrospinal fluid, homogenization of the sample is not possible in nail specimens. Fungi may be in one nail sample and may not be in the other. Amichai B et al. (19) showed that, of the 147 cases of clinical onychomycosis initially thought to be negative, 138 (94%) were later found to be positive after up to four consecutive sets of laboratory mycological investigations. Therefore, he suggested starting systemic antifungal treatment in patients with suspected fungal infections, even if they have negative laboratory fungal examinations which may be useful in some cases. We reinforced his findings showing equal cure rate in microscopy positive samples and microscopy negative samples. Histopathological periodic acid-schiff stains (PAS) of nail clippings found to be more sensitive (75% positivity) than direct microscopy (53%) and culture (35%) in recent studies (20–22). Information concerning the vitality of the fungi and accurate identification of the specific pathogen is not available through this investigation alone, mycological culture continues to remain the indisputable ‘‘gold standard’’ of mycological diagnostics. Onychomycosis, though considered a cosmetic problem, is a debilitating disease with immense negative physical and psychological impact. Secondly, no spontaneous clearing is known to occur. Moreover, untreated patients can act as a reservoir for family contacts and can contaminate communal bathing places. In the elderly, it can lead to cellulitis, while in diabetic patients, it carries the risk of development of diabetic foot. All these factors necessitate treatment for onychomycosis (23). In our study, we showed that both itraconazole and terbinafine in pulse therapy have 54–65% clinical cure rate on NDM onychomycosis. This further proved those current newer antifungal are less effective in treating NDM onychomycosis. However, duration of therapy and cure rate was comparable to dermatophytes. Both drugs were safe, except for nausea in two patients who had terbinafine and worsened gastritis in two patients with itraconazole. Persistent predisposing factors, such as frequent immersion of hands in water in house wives, contact with mud and soil in farmers and manual laborers, that are unavoidable habitual and occupational hazards may further add to the drug unresponsiveness. Same patient having some nails cured, while other nails not cured is unexplainable by that speculation alone. The gold standard treatment for onychomycosis is basically systemic. Combination with topical agents, such as nail lacquer


and/or chemical nail avulsion, produces better results than systemic treatment alone. Topical treatment as monotherapy is not efficient, excluding minor cases. NDM are often difficult to eradicate and usually require a combination of approaches to achieve success (24). Although itraconazole have a broad spectrum of activity, there is paucity of data from larger clinical trials regarding its efficacy in NDM onychomycosis and treatment regimes are not well standardized. Tosti et al. (25), in their study of 59 cases of NDM, reported better cure rates with a combination of topical treatment and surgical avulsion a (60%) compared to monotherapy with terbinafine or itraconazole alone (20% Acremonium species, 29% Fusarium and 42% Scopulariopsis brevicaulis). Aspergillus was the only exception that responds well to systemic therapy with either terbinafine or itraconazole (8,26). Treatment of other molds is often challenging and may require combination of oral, topical and surgical methods (27,28). Therapy differs in terms of clinical presentation as well; SWO can be treated with abrasion of nail surface followed by topical therapy with nail lacquers. Systemic therapy should be added if SWO is originating from proximal nail fold. DLSO can be treated with itraconazole or terbinafine therapy. In conclusion, NDM onychomycosis was the prevailing fungi in Sri Lanka, followed by candida spp, whereas dermatophytes made up only 10.1%. With itraconazole pulses 68.22% Aspergillus spp, 50% Fusarium spp and 84.6% Penicillium spp showed clinical cure, while terbinafine pulses cured 55.0% Aspergillus spp and 50% Fusarium spp. No statistically significant difference was detected between the two regimens for the clinical cure and recurrence rate. However, better clinical cure was observed with itraconazole pulses (65.1%) than with terbinafine pulses (54.64%), and recurrence was higher with itraconazole (6.5% vs 4.1%). Since both regimens were only partially effective on NDM onychomycosis showing a clinical cure of 54–65%, future research should focus on searching more effective antifungal for the treatment of nondermatophyte onychomycosis.

Declaration of interest The authors declare that there is no conflict of interests.

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