Molecular characterization and antifungal activity against non-dermatophyte molds causing onychomycosis

Onychomycosis is a fungal disease that caused by different types of fungi. Non-dermatophyte molds are a large saprophytic fungi group that live in nature and could affect traumatic nails. The aim of this study was to identify non-dermatophyte molds causing onychomycosis and evaluation of several antifungal activities against the isolates. The samples consisted of 50 non-dermatophyte molds isolated from patients with onychomycosis confirmed by direct and culture examination fungal. DNA was extracted, amplified, and sequenced. Disk diffusion method was used to evaluate itraconazole, fluconazole, ketoconazole, terbinafine, posaconazole, and econazole activity against the isolates. The species identified as: Aspergillus flavus 22 (44%), A. niger 12 (24%), A. fumigates, 3 (6%), A. sydowii 3 (6%), A. terreus 1 (2%), Penicillium commune 2 (4%), P. glabrum 2 (4%), P. chrysogenum, 1 (2%), Fusarium solani 3 (6%) and F. thapsinum 1 (2%). Most of the samples were sensitive to terbinafine, itraconazole, and econazole and 94% of the isolates were resistant to fluconazole. This study showed that Aspergillus species were the most common cause of non-dermatophyte mold onychomycosis and fluconazole was the most resistant antifungals. Care must be taken to choose the appropriate antifungal drug for a better cure.

www.nature.com/scientificreports/ onychomycosis: allylamines such as terbinafine, azoles such as itraconazole, morpholines, hydroxypyridinones (ciclopirox), and echinocandins 16 . The diagnosis of onychomycosis is conventionally direct microscopy and fungal culture; however, culture-based detection methods may take a few weeks for definite identification. Molecular method techniques using polymerase chain reaction (PCR) assay provide a rapid, stable, and accurate alternative for identifying pathogenic fungi in the affected nail samples of patients with onychomycosis 17 .
Onychomycosis's causative agents may vary depending upon geographic or temporal distribution, and inadequate treatment may lead to infection resistance and recurrence. Proper clinical diagnosis, laboratory workup, and adequate antifungal therapy are thus the standard of care for nails infections, so this study was aimed to find out the pattern of NDMs as causative agents of onychomycosis based on DNA sequence analysis of internal transcribed spacer (ITS) ribosomal DNA (rDNA) and evaluation of antifungal drug activities by disk diffusion methods.
The results of culture and sequencing indicate that the three genus of Aspergillus, Penicillium, and Fusarium were identified equally in both methods. Two species of flavus and niger were identified equally at the species level, but the other unknown species could identify by the sequencing method (Fig. 2).
The antifungal drugs patterns (susceptible, intermediate, and resistant) against NDMs is shown in Table 2. The highest sensitivity was observed in terbinafine, itraconazole, and econazole, respectively. Of all these sensitive isolates, two cases were intermediate to terbinafine and econazole, and 1 case was resistant, and 1 case was intermediate to itraconazole. Forty-two isolates (84%) were sensitive to posaconazole, and the remaining were   (Fig. 3). Based on the criteria of sensitivity and resistance, the highest resistance was observed in fluconazole, and it was statistically significant among other drugs (p-value < 0.001).
It is noteworthy that 47 out of 50 cases (94%) were resistant to fluconazole, and only 1 case was sensitive to this drug, and the remaining two were intermediate.
In three genus, 1 Aspergillus, 2 Penicillium, and 2 Fusarium isolates were resistant to posaconazole and fluconazole. There was only one isolate resistant to itraconazole, which was related to A. flavus species (Table 2).
Also, the resistance samples to posaconazole compared to itraconazole and ketoconazole were significant. The p-value in both was 0.036, so the resistance of the samples to posaconazole was higher than itraconazole and ketoconazole.   www.nature.com/scientificreports/ The most effective drugs were terbinafine, itraconazole, and econazole that 96% of the samples were sensitive. The next most effective drugs were ketoconazole and posaconazole, which 84% and 72% of the samples were sensitive to them, respectively. Sensitive samples to econazole, itraconazole, and terbinafine compared to ketoconazole were statistically significant with a p-value of 0.001, so samples' sensitivity to econazole, itraconazole, and terbinafine were higher than ketoconazole. There was no significant difference in the samples' sensitivity to ketoconazole, econazole, itraconazole, and terbinafine compared to posaconazole (p-value > 0.05). The susceptibility of most frequent species (A. flavus and A. niger) to all drugs except ketoconazole and fluconazole was the same (p-value > 0.05), but ketoconazole was found to be more effective in flavus species than niger species (p-value < 0.001). Both species were determined to have a fluconazole-resistant ratio.

Discussion
Onychomycosis is one of the most common nail infections in which dermatophytes, yeasts, and non dermatophyte molds have been incriminated as etiological agents. According to the latest reports, the global prevalence of onychomycosis is nearly 2-9% 7 . Onychomycosis often was mimicked with psoriasis of the nail, eczema, bacterial infections, and contact dermatitis 18 . So, correctly determining the etiologic agents of onychomycosis is important to provide a baseline for administering appropriate antifungal therapy and identifying the source of infection, hence facilitating prevention measures 19 .
At the moment, traditional mycology remains the gold standard for diagnosing NDMs onychomycosis. This includes obtaining positive results from KOH and culture. The culture method, although it demonstrates the viability and allows the identification of isolated fungi but it is time-consuming, and due to the diversities and similarities among different species, morphological methods are not specific enough. It appears that a more accurate method is critical to differentiate various members of the genus 20 .
In addition to standard procedures, the use of molecular biological techniques such as polymerase chain reaction (PCR), followed by sequencing of appropriate targets, has important implications for understanding these pathogenic fungi 21 . Scopulariopsis brevicaulis, Fusarium species, Aspergillus species, Neoscytalidium dimidiatum, and Acremonium species are the most common causative agents of onychomycosis in the world that we could not isolate some of them such as S. brevicaulis, N. dimidiatum or Acremonium spp. 13 . Comparison of culture and molecular methods indicated that Aspergillus, Penicillium, and Fusarium genus were identified in both methods. Using molecular methods increased the sensitivity and specificity of identification of microorganisms that caused onychomycosis as our 16 unknown isolates were identified by this method as followed: A. fumigatus (n = 3), A. sydowii (n = 3), A. terreus (n = 1), Penicillium commune (n = 2), P. glabrum (n = 2), P. chrysogenum (n = 1), Fusarium solani (n = 3) and F. thapsinum (n = 1).
In this study, A. flavus was responsible for approximately 44% of all the samples and that this data was agree with Nouripour-Sisakht et al. 22 , Chadeganipour et al. 23 and Halvaee et al. 24 , they reported that 77.3%, 66% and 60.9% of their isolates were A. flavus, respectively. The high frequency of nail infections due to A. flavus can be attributed to the fact that dry climate in Iran that favors the growth of thermo-tolerant fungi like A. flavus and this species is one of the most frequent Aspergillus species isolated from the environment 25,26 . This genus is one of the common pathogenic fungi in humans 27  Penicillium commune and P. glabrum that identified were reported for the first time from onychomycosis patients in Iran but also those fungi have been isolated from environmental area of Mazandaran province in north of Iran previously and, identified by beta-tubulin gene sequencing method 29 . It is hard to identify these organism by conventional methods maybe for this reason there was less reports regarding these isolates from onychomycosis around the world. Penicillium chrysogenum as pathogenic fungi is involved in developing skin diseases such as onychomycosis and invasive diseases 30 .
Treatment is necessary for onychomycosis to avoid serious consequences such as secondary bacterial infections, complete nail dystrophy, and cosmetic reasons 31 . It is including use topical and oral agents and physical www.nature.com/scientificreports/ treatments 32 . Drug choice depends on several things such as the severity of nail damage, type of organism, adverse effects, and success or failure of previous treatments 33 . Treatment in NDMs onychomycosis is dependent on fungal species that caused infection and their susceptibility to antifungals prescription because different fungi have different responses to drugs. On the other hand, many of the selected therapies were failed because of increasing the prevalence of drug resistance related to the fungi genetic factors and the type of antifungals or host conditions 33 .
Drug susceptibility tests have several benefits, such as preventing long-term, unnecessary, and even high-risk drug use and reducing treatment costs 34 .
The disk diffusion method is a simple, inexpensive, and rapid method that qualitatively demonstrates the antifungal drugs' effectiveness 35 . In our study, terbinafine, econazole, and itraconazole were identified as the most effective antifungal drugs against onychomycosis caused by NDMs agents. In fact, out of the 50 isolates, 96% of them were sensitive to these three antifungals.
Except for several cases of Fusarium solani and Aspergillus flavus all the isolates were sensitive to terbinafine, econazole and itraconazole.
As shown in our in vitro study, terbinafine and itraconazole were the best candidates for NDM onychomycosis treatment, but it needs more details about in vivo data analysis. One reason for the good effects of terbinafine is its appropriate penetration into the nail matrix 36 .
As we showed in this study, all Aspergillus species were sensitive to terbinafine, and this result agreed with Gupta et al. 13 and Trovato et al. 37 in Italy and Haghani et al. 38 and Xu et al. 25 in Iran. In other study in Iran reported that some Aspergillus species were resistance to terbinafine and this result was not agree with our study 39 .
A noteworthy point in this study was the high resistance of isolates to fluconazole (94%). All species of Aspergillus were resistant to fluconazole. Our results agreed with Tsang et al. 40 study and were in contrast with Haghani et al. 38 that reported Aspergillus terreus was sensitive to fluconazole. So, fluconazole is not suitable to cure onychomycosis causing by NDMs but is commonly prescribed in combination with the other antifungals such as itraconazole and terbinafine cases of moderate to severe nail involvement. The results in Falahati et al. study showed that the clinical isolate of A. clavatus resistant to the wide spectrum of antifungal drugs such as terbinafine, posaconazole, voriconazole, etc. but sensitive to itraconazole 39 . Due to the side effects and high resistance of NDMs to fluconazole, drug susceptibility testing is important 31 .
It is necessary to mention that two isolates of Fusarium solani were resistant to all five antifungal drugs, except each one of them was sensitive to econazole and itraconazole separately. Bueno et al. also reported Fusarium as resistant fungi to antifungal drugs in his study 41 . Although Fusarium had less frequency than the other genus in our study, special attention should be given to these genus because of their antifungal resistance and keratinolytic activity that helped Fusarium to use nails as a source of nutrition 42 .
The high prevalence rate of onychomycosis with different fungi groups involving etiological agents suggests that more studies should be done on the prevalence. This is for better understanding these issues and could cause developing better preventive procedures and reducing treatment costs. Also, in vivo studies are needed to confirm the effects of drugs in order to find a suitable drug to treat these cases of onychomycosis.

Conclusion
Onychomycosis is a common multifactorial disease of the nails. The prevalence of fungal nail infections is affected by age, lifestyle, and some underlying diseases. A global outlook on the treatment of onychomycosis provides a framework of success for the clinician. Healthcare could be more effective if physicians prescribe the most effective antifungals for the treatment of onychomycosis.

Materials and methods
Collection of nail samples. Fifty NDMs onychomycosis cases among 300 clinically suspected patients with onychomycosis referred to medical mycology laboratory enrolled in this study. Patients who had a previous history of antifungal therapy were excluded from the study. Small pieces of the nail debris were collected from the affected area using sterile nail clippers and scalpel.

Mycological examination.
A part of nail scrapings was immersed in a drop of 20% KOH over a slide and kept in a wet chamber at room temperature for direct microscopy examination.
The other parts of the nail samples were simultaneously inoculated on Sabouraud's dextrose agar (SDA) (Merck, Darmstadt, Germany), SDA containing chloramphenicol (0.05 mg/l), with and without actidione (500 mg/l). The cultures were incubated at 28 °C for 1-2 weeks. The plates were examined twice weekly for any growth. No growth at the second week was considered as negative. A broad septate/non septate hyphae detection in the direct exam along with positive culture (saprophyte colony growth) was considered as NMD onychomycosis. Genus and species of fungi were identified by colony morphology and microscopic examination by using teas mounts and slide culture. Diagnosis of NDM onychomycosis was made on the basis of the following criteria: (1) nail abnormalities; (2) positive KOH, (3) isolation in culture, (4) repeated isolation in culture (5) failure to isolate a dermatophyte in culture; and in suspicious cases we checked growth of more than 5 colonies of the same mold in at least 2 repeated nail samplings 43,44 . Preparation of genomic DNA. Total cellular DNA was extracted from a small amount of mycelium cultured on Sabouraud agar slants, by the rapid mini-preparation method 45 . Briefly, to a 1.5 ml eppendorf tube containing 500 µl of lysis buffer (400 mM Tris-HCl [pH = 8], 60 mM EDTA pH [pH = 8], 150 mM NaCl, 1% [w:v] SDS), a small portion of mycelium grown on SDA in primary culture was added using a sterile toothpick, with which the mycelia were disrupted. The tube was then left at room temperature for 10 min. After adding 150 µl of Table 3. Inhibition zones diameters (mm) of antifungal tablets examined in this study after inoculation of non-dermatophytic mold suspension. DD = doses dependent.