The epidemiology and etiology of onychomycosis in 2 laboratory centers affiliated to Tehran university of medical sciences during 2019-2020.

Background and
Objectives: Onychomycosis is caused by dermatophyte species, non-dermatophyte moulds (NDMs), and accounts for roughly 50% of all nail diseases. As the prevalence of onychomycosis is increasing, new epidemiologic documents may help with treatment and prevention. The present investigation aims to determine the epidemiological profile of onychomycosis in 2 mycology laboratories. Materials and
Methods: A cross-sectional study conducted during eight months (2019-2020) on 169 patients with positive nail mycology tests referred to two mycological laboratory centers affiliated with Tehran University of Medical Science. The nail clippings were examined by direct smear and culture. Also, molecular assays were performed if needed.
Results: 10% of nail lesions referred to Razi Hospital (RH), and 30% of nail lesions referred to TUMS mycology laboratory were positive. Middle age (40-60) suffer more from onychomycosis. Aspergillus flavus, Trichophyton mentagrophytes, and Candida albicans were the most common etiologic agents in each of the three main classes of fungi causing onychomycosis. Females were more infected. NDMs were the predominant etiologic agents, and toenails were the most common site of onychomycosis.
Conclusion: The pattern of etiologic agents and clinical signs of onychomycosis differs according to geographical region and age, so repeated epidemiological surveys of onychomycosis seem to be fundamental.

INTRODUCTION

The term “onychomycosis” is originally a Greek word derived from the terms “onyx,” meaning nail, and “makes,” meaning fungus (1–3). Onychomycosis is the most common disease affecting the nail unit and accounts for at least 50% of all nail disease (3). It can cause by dermatophytes, non-dermatophyte moulds (NDMs), and yeasts. Based on recent published epidemiologic researches, the prevalence of onychomycosis is about 5.5% globally (2, 4). Because the nail unit does not have effective cell-mediated immunity, it is vulnerable to fungal infection (3). Studies demonstrated that toenail involvement is the more common clinical form of onychomycosis in males, while Candida fingernail type is more common in females (5–6). The other predisposing factors are fungal infection elsewhere on the body (in particular, tinea pedis), chronic paronychia, previous onychomycosis, wearing of occlusive and tight shoes, hyperhidrosis, participation in sports or fitness activities, nail trauma, poor nail grooming, use of commercial swimming pools, communal bathing, living with family members suffering from fungal infection, poor health, genetic factors, immunodeficiency (in particular, acquired immune deficiency syndrome and transplant patients), diabetes mellitus, obesity, Down syndrome, psoriasis, smoking, peripheral vascular disease, venous insufficiency, hallux valgus, and asymmetric gait nail unit syndrome (2, 4, 5–7). It should be noted that the etiologic agent of onychomycosis varies in different countries and different provinces of a country like Iran. The present study tries to identify the etiologic agents of onychomycosis and assess the current epidemiology of this infection based on age, gender, and site of illness in patients referred to a mycology hospital center and a faculty laboratory in Tehran province, Iran.

MATERIALS AND METHODS

Ethics statement.

The Research Ethics Committee approved the study of Tehran University of Medical Sciences (the number of Ethics Committees protocol: IR.TUMS.SPH.REC.1398.197). The project was founded on the ethical principles and the national norms and standards for conducting Medical Research in Iran.

Study design, patients and setting.

The experimental interventional study was carried out for eight months from 2019.07.10 to 2020.03.09. Sampling was arranged on patients who presented nail changes compatible with a clinical diagnosis of onychomycosis, including paresthesia, pain, onycholysis, nail discoloration, brittleness, subungual hyperkeratosis, splitting of the nail plate, and nail plate destruction. The exclusion criteria were patients using topical and/or systemic antifungals at the time of sampling or up to 15 days before collecting the specimen and patients whose clinical samples were insufficient for complete analysis. Nail scrapings were gathered from outpatients referred to the mycology laboratories of Razi Hospital (RH) and Tehran University of Medical Science (TUMS). Both mycology laboratories were not private and were subdivisions of TUMS. The demographic and clinical data were documented in the patients’ sheets.

Culture and phenotypic examination.

One sample was used to perform a potassium hydroxide (KOH) mount. The second was inoculated in three separate areas into Sabouraud dextrose agar (SDA-Merck, Germany) supplemented with 0.5% chloramphenicol and incubated at 26°C for 20 days and checked daily. Any growth obtained was identified by its characteristics include colony morphology, growth rate, and colony pigmentation. For obtaining pure single colonies and preliminary identification of yeasts, the grown yeast isolates were sub-cultured on CHROMagar Candida medium (CHROMagar, Paris, France). Identification of isolated dermatophytes and NDMs was made based on colonial morphology and microscopic characteristics using lactophenol cotton blue and slide culture. The diagnosis was based on similar growth in three separated sample growth and positive microscopic examination for identification saprophytic fungi from environmental saprophytic contamination. In this study, the correct determination of samples that were not detectable at the species level by mycological techniques, DNA sequencing was performed.

Molecular technique: DNA extraction, PCR conditions and sequencing.

DNA was extracted by using the High Pure PCR Template Preparation kit (Roche, Germany) according to the manufacturer’s recommended instructions. PCR assay was performed using the three μL of test sample as a template, in a total volume of 25 μL (1 μL of each of forward and reverse primers, ten μL of PCR MasterMix (Amplicon, Denmark) and, nine μL of deionized distilled water based on the following protocol: 10 min of primary denaturation at 95°C, 40 cycles of denaturation for 20 sec at 95°C, annealing for 20 sec at 62°C, an expansion for 20 sec at 72°C, and a final extension for 5 min at 72°C. Eventually, the products were run on a 2% agarose gel. The amplification of Aspergillus isolates was conducted by the β-tubulin primers (BT-forward (5′-GGTA ACC AA ATCGGTG CTGCTTTC-3′) and BT-reverse (5′-ACCCTCAGTGTAGTGA CC CTTG-GC-3′). Also, other fungal species were identified to the species level using the universal primers: ITS1 (5′TCC GTA GGT GAA CCT GCG G 3′), and ITS4 (5′TCC TCC GCT TAT TGA TAT GC 3) (Life Technologies, Barcelona, Spain). The PCR products were examined by staining with DNA-safe stain and electrophoresis on 1.5% agarose gel. The PCR products were subjected to sequencing and analyzed using the MEGA7.0.21 software.

RESULTS

In the present study, in the mycology laboratory, RH nail mycology requests calculated 10.5% of all mycology tests. Also, in the mycology laboratory of TUMS, nail mycology requests calculated 34.7% of all mycology tests. Furthermore, positive results for onychomycosis in RH and in TUMS were estimated at 41.9% and 30% of total nail mycology tests, respectively. The woman to men ratio in RH was 5:1(89/16) and in TUMS was 2:1(85/43). The average age of referred patients to two investigated centers was about 40–50 years that means onychomycosis is a middle-aged disease in both genders (Table 1).

Table 1.

Relative and cumulative frequency of onychomycosis based on gender and age.

Gender	Male		Female		Total No
Age	Per	No	Per	No
0–17	1.8	3	0.6	1	4
18–25	0	0	7.1	12	12
26–35	3.6	6	8.9	15	21
36–45	5.3	9	13.6	23	32
46–60	11.8	20	18.9	32	52
60≤	14.2	24	14.2	24	48
Total	36.7	62	63.3	107	169

Abbreviations: Per: percent, no: number

A total of 169 patients with onychomycosis claimed for culture. The results indicate that laboratory confirmation was reached through 121 (71.6%) positive direct smear and culture tests, 42 (24.8%) positive direct smear and negative culture tests, and 6 (3.6%) negative direct smear and positive culture tests. The age range of patients was between 2 and 87 years (mean age, 46.7 years), 62 male (13 with fingernail involvement, 49 with toenail involvement) and 107 females (56 with fingernail involvement, 51with toenail involvement) (Table 1). Three patients (1.8%) had fingernail and toenail onychomycosis simultaneously, and the etiologic agents of fingernail and toenail involvement in 2 of them (66.6%) were the same.

Seventy-five non-dermatophyte spp. (44.4%), fifty-seven Candida spp. (33.7%), and thirty-seven dermatophyte spp. (21.9%) were isolated from nail infection in this investigation.

The anatomical sites of onychomycosis in the present study were toenails (n-=100, 59.2 %) and fingernails (n=69, 40.8%), respectively. Also, 56 (81.1%) of fingernail onychomycosis belonged to females, while 13 (18.9 %) of fingernail lesions belonged to men. A statistically significant relation was found between gender and the site of infection (P-value<0.05) (Table 2).

Table 2.

Frequency of etiologic agents of onychomycosis based on gender and the site of involvement

	Gender								Total
	Female				Female
	Fingernail		Toenail		Fingernail		Toenail
Yeasts	10	5.91%	6	3.55%	36	21.3%	5	2.95%	57	33.7%
Dermatophytes	1	0.591%	23	13.6%	2	1.183%	11	6.50%	37	21.8%
Moulds	2	1.183%	20	11.8%	18	11.83%	35	20.71%	75	44.4%
Total	13	7.7%	49	29%	56	33.1%	51	30.%	169	100%

The most frequent toenail onychomycosis was seen in 46–60 and over 60-year-old age groups equally (13.6%). The lowest frequency of onychomycosis is due to dermatophytes in the 0–17-year age group (0%). Also, the highest frequency of onychomycosis due to dermatophyte was found in the 46–60-year age group (47.3%), and the highest frequency of onychomycosis due to yeasts was found in the over 60 years (31.6%) age group. Furthermore, the highest frequency of onychomycosis due to NDMO was found in the 36–45- and the 46–60-year age group (24.3%) equally (Table 3).

Table 3.

Relative and cumulative frequency of different etiologic agents of onychomycosis based on age groups

Etiologic agent	Yeasts		Saprophytic fungi		Dermatophytes		Total
Age groups (yrs)	Number	%	Number	%	Number	%	Number	%
0–17	3	75	1	25	-	-	4	100
18–25	1	8.3	9	75	2	16.7	12	100
26–35	7	33.3	11	52.4	3	14.3	21	100
36–45	12	37.5	19	56.25	1	6.25	32	100
46–60	16	30.8	18	34.6	18	34.6	52	100
≥60	18	37.5	17	35.4	13	27.1	48	100
Total	57	33.7	75	43.8	37	22.5	169	100

There was a significant relationship between the etiologic agent of onychomycosis and the site of infection (P-value <0.00006) (Table 4).

Table 4.

Relative and cumulative frequency of different etiologic agents of onychomycosis based on infected situation

Site of infection	Onychomycosis etiologic agents			Total
	Non-dermatophyte moulds	Dermatophyte	Yeast
Fingernail	20 (29%)	3 (4%)	46 (67%)	69 (100%)
Toenail	55 (55%)	34 (34%)	11 (11%)	100 (100%)
Total	75 (43.8%)	37 (22.1%)	57 (33.7%)	169 (100%)

Dermatophytic onychomycosis was more frequent in males (64.8%) than females (35.2%), and there was a significant relationship between the patient’s gender and dermatophytic onychomycosis involvement (P-value <0.05), but no significant correlation was found.

Table 5 shows the relative and cumulative frequency of etiologic agents of onychomycosis diagnosed by mycological techniques in the present study. Also, for a correct determination of isolates which were not detectable by mycological techniques, DNA sequencing was performed. All of the sequences had been deposited in GenBank under the accession number reported in Table 6.

Table 5.

Relative and cumulative frequency of etiologic agents of onychomycosis diagnosed by mycological techniques

Fungal pathogen	Fungi	Number	Percent(in each fungal group)	Percent(in total)
Non-dermatophytic moulds (n=75, 44.4%)	Aspergillus flavus	30	40	17.8
	Aspergillus niger	20	26.7	11.8
	Aspergillus terreus	7	9.3	4.1
	Fusarium spp.	4	5.3	2.3
	Cladosporium spp.	2	2.6	1.2
	Penicillium spp.	3	4	1.8
	Mucor spp.	1	1.3	0.6
	Black fungus	1	1.3	0.6
	Alternaria spp.	1	1.3	0.6
	Saprophytic sterile mycelium	6	8	3.5
Dermatophytes (n=37, 21.9%)	Trichophyton mentagrophytes	8	21.6	4.7
	Trichophyton rubrum	6	16.2	3.6
	Trichophyton verrucosum	3	8.1	1.8
	Trichophyton tonsurans	3	8.1	1.8
	Trichophyton violaceum	1	2.7	0.6
	Microsporum canis	1	2.7	0.6
	Microsporum audouinii	1	2.7	0.6
	Dermatophyte spp.	14	37.8	8.2
Yeasts (n=55, 33.7%)	Candida albicans	17	30.9	10.1
	Candida parapsilosis	15	27.3	8.8
	Candida glabrata	8	14.5	4.7
	Candida tropicalis	2	3.6	1.2
	Trichosporon spp.	2	3.6	1.2
	Rodoturolla spp.	2	3.6	1.2
	Candida spp.	9	16.4	5.3

Table 6.

The results of molecular identification and GenBank accession numbers of DNA sequences included in this study

Isolate	Molecular identification (IT’S gene)	GenBank accession number	Isolate	Molecular identification (ITSa gene )	GenBank accession number
SUB10203970 seq1	Neoscytalidium dimidiatum	MZ882261	SUB10203970 seq26	Cladosporium pseudocladosporioides	MZ882286
SUB10203970 seq2	Candida albicans	MZ882262	SUB10203970 seq27	Trichophyton mentagrophytes	MZ882287
SUB10203970 seq3	Candida parapsilosis	MZ882263	SUB10203970 seq28	Penicillium glabrum	MZ882288
SUB10203970 seq4	Candida albicans	MZ882264	SUB10203970 seq29	Mucor circinelloides	MZ882289
SUB10203970 seq5	Penicillium glabrum	MZ882265	SUB10203970 seq30	Aspergillus flavus	MZ882290
SUB10203970 seq6	Fusarium fujikuroi	MZ882266	SUB10203970 seq31	Aspergillus flavus	MZ882291
SUB10203970 seq7	Aspergillus flavus	MZ882267	SUB10203970 seq32	Trichophyton mentagrophytes	MZ882292
SUB10203970 seq8	Alternaria infectoria	MZ882268	SUB10203970 seq33	Trichophyton tonsurans	MZ882293
SUB10203970 seq9	Trichophyton tonsurans	MZ882269	SUB10203970 seq34	Rhodotorula mucilaginosa	MZ882294
SUB10203970 seq10	Trichophyton rubrum	MZ882270	SUB10203970 seq35	Trichophyton mentagrophytes	MZ882295
SUB10203970 seq11	Trichophyton rubrum	MZ882271	SUB10203970 seq36	Trichosporon asahii	MZ882296
SUB10203970 seq12	Aspergillus niger	MZ882271	SUB10203970 seq37	Fusarium fujikuroi	MZ882297
SUB10203970 seq13 T	richophyton mentagrophytes	MZ882273	SUB10203970 seq38	Fusarium fujikuroi	MZ882298
SUB10203970 seq14	Penicillium polonicum	MZ882274	SUB10203970 seq39	Trichophyton mentagrophytes	MZ882299
SUB10203970 seq15	Aspergillus niger	MZ882275	SUB10203970 seq40	Trichophyton rubrum	MZ882300
SUB10203970 seq16	Candida albicans	MZ882276	SUB10203970 seq41	Trichosporon asahii	MZ882301
SUB10203970 seq17	Aspergillus flavus	MZ882277	SUB10203970 seq42	Trichophyton mentagrophytes	MZ882302
SUB10203970 seq18	Candida parapsilosis	MZ882278	SUB10203970 seq43	Trichophyton rubrum	MZ882303
SUB10203970 seq19	Cladosporium herbarum	MZ882279	SUB10203970 seq44	Trichophyton mentagrophytes	MZ882304
SUB10203970 seq20	Candida parapsilosis	MZ882280	SUB10203970 seq45	Fusarium fujikuroi	MZ882306
SUB10203970 seq21	Aspergillus flavus	MZ882281	SUB10203970 seq46	Trichophyton rubrum	MZ882306
SUB10203970 seq22	Candida parapsilosis	MZ882282	SUB10203970 seq47	Trichophyton mentagrophytes	MZ882307
SUB10203970 seq23	Rhodotorula mucilaginosa	MZ882283
SUB10203970 seq24	Candida tropicalis	MZ882284
SUB10203970 seq25	Candida parapsilosis	MZ882285

DISCUSSION

Treatment of onychomycosis needs a long-time treatment protocol, and an accurate diagnosis is essential. Precise diagnosis is made on the basis of clinical manifestation and laboratory confirmation using microscopy, culture, and molecular tests (2). Also, laboratory confirmation of the clinical diagnosis of onychomycosis prior to initiating treatment is cost-effective and is recommended (2). Treatment assessment, including drug of choices, device treatments, and treatment duration, depends on accurate identification of etiologic agents of onychomycosis, too. In this study, NDMs were the predominant etiologic agent of onychomycosis, followed by yeasts and dermatophytes. But onychomycosis etiologic agent in a study conducted by Aghamirian was dermatophytes, yeasts, and saprophytic moulds, respectively (1). Also, in another study by Halvaee, dermatophytes were diagnosed as the most common etiologic agents of onychomycosis, followed by yeast and NDMO (2).

Previously a study in the Khuzestan province of Iran hypothesized that the causative agents of onychomycosis have shifted from dermatophytes to yeasts (3). But a meta-analysis that reviewed all published studies about the epidemiology of onychomycosis from Iran did not confirm it (4). This difference may be due to the predominant middle-aged population group in this study.

It should be noted in the present study onychomycosis caused by NDMs in toenails was more frequent than fingernails. This finding is similar to the results of previous studies (2, 7–9). The most frequent NDMs isolated from nail samples in the present study were A. flavus (40%) and A. niger. The finding is consistent with the results of other studies in Iran (10), Nepal (11), and India (12). Studies showed that A. flavus was the most distributed species among genus Aspergillus in indoor and outdoor environments in Iran. More distribution of A. flavus in the environment can facilitate exposure and increase the risk of colonization and infection with this species (13). In this study, a case of toenail onychomycosis caused by Neoscytalidium dimidiatum was reported and confirmed by molecular sequencing (The obtained sequence with accession number MZ3377100 was 100% compatible with Neoscytalidium dimidiatum clone URF_Pt01) as the first one in Iran.

Furthermore, in this study, the most frequent isolated dermatophytes were T. mentagrophytes (21.6%) and T. rubrum (16.2%), which was in accordance with the results of studies previously conducted in Iran (2, 14), Germany (15), and Canada (16). Although, results of studies conducted in Tehran (14), and India (17) indicated that the most frequent isolated dermatophyte moulds were T. rubrum, T. mentagrophytes, T. violaceum, and Epidermophyton floccosum, subsequently. Also, C. abidance (30.9%) and C. parapsilosis (27.3%) subsequently were the most frequent yeasts isolated of infected nails. This finding was similar to other studies (18–20).

In the present investigation, the prevalence of onychomycosis was 41.9% in RH and 30% in TUMS mycology laboratories. The difference between the two studied centers might be due to the patients referred to each center because one was a dermatology hospital and the other was a single-specialized laboratory center. The prevalence of onychomycosis in both centers was lower than those previously reported in Dakar (Senegal) (48.4%) (21), Ethiopia (60.4%) (22), and North-East of Iran (56.8%) (23), and higher than those previously reported in Isfahan (13.1%) (24), South Greece (27.99%) (25), Northwestern Greece (28.9%) (26), and similar to those previously reported in other provinces of Iran such as Khuzestan (35.6%) (3), Tehran (39.6%) (27) and Qazvin (40.2%) (1).

In the present survey, the most frequency of onychomycosis in both genders has belonged to those over 45 years of age. The finding was similar to the results of studies conducted by Halvaee (40–60-year age group) (2), Rafat (>50 years in both genders) (4), and Aghamirian (40–49 years in both genders) (1). Different researches show onychomycosis is much more common in adults than in children, and the prevalence increases with age (2, 4). A prevalence of 0.4% in children of North America was reported for onychomycosis (28), whereas the prevalence may be as high as 33% in the elderly (> 60 years of age) (2). Also, in the present study, Fingernail onychomycosis was more common in females than men significantly. It was compatible with the results of a meta-analysis conducted by Rafat in Iran (4). Also, in the present study, 24.8% of specimens did not grow that may be due to unknown factors like washing the lesions, insufficient samples, or the use of antifungal drugs.

Furthermore, the results showed that females were more affected by onychomycosis than males that were compatible with the results of previous studies (2, 6). A higher ratio of women may be due to more hand eczema in the industrial cities in women (29), whom more refer to dermatology hospitals.

Regarding the fact that in the present study, the most frequent rate of onychomycosis was seen in 40–60-year-old people, the higher frequency of onychomycosis caused by NDMs may be due to the presence of predisposing factors such as occlusive and tight shoes footwear and trauma in this age group. This finding is similar to the results of studies conducted by Halvaee (2), Hilmioğlu (7), Moreno (8) and Gianni (9). More toenail involvement may be due to more trauma exposure in toenails, making them vulnerable to saprophytic fungi. Also, low hygiene in toenails than fingernails, the bareness of hands, high humidity, and more soil exposure are other reasons for this finding.

Also, in the present study, a 2.5-year patient with fingernail onychomycosis due to Candida species was found, which may be due to finger sucking or anal scratching. In the present study, the most common site of onychomycosis was the toenail.

CONCLUSION

This study is an advanced cause of the toenail problem that is more common in the middle-aged population. This finding is essential for health care that might be ignored besides more critical health problems like hypertension, diabetes, senile arthritis in this age group. Because toenails are an exceptional part of our beauty, the development of preventive and educational strategies about onychomycosis can help prevent nail dystrophy, the spread of infection and more flexibility in walking, running and other fast movements that depend on the precise balance of the weight tolerance. The present study started in the fall of 2018 and ended in May of 2019, and therefore the SARS-COVID-19 restrictions in the two last months of sampling led to a decreased number of patients referred to mycology laboratories.