الفهرس 
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Abstract
Onychomycosis is a significant worldwide disease which represents 20% to 60% of all nail conditions. Therefore, it is essential to provide available and good laboratory methods for rapid and precise identification of fungi causing onychomycosis to apply appropriate treatment and preventive measures.
There are three conventional tests used in the Outpatient Clinic; potassium hydroxide (KOH) direct microscopic technique, pathological examination of nail plate tissue by periodic-acid Schiff (PAS), and fungal culture of nail plate debris. These methods are commonly used but have many disadvantages. For example, direct microscopy of KOH mounts requires experienced investigators and may give false-negative results in 5–15% of cases. PAS staining couldn’t confirm the causative organism or its viability. Fungal culture is a time-consuming procedure with higher percentage of false negative results (30–50%).
Therefore, more rapid and sensitive non-conventional methods for the identification of fungal species are necessary e.g microscopic examination by fluorescent staining and molecular diagnostic methods.
The aim of this study was to highpoint the variable risk factors contributing to the occurrence of onychomycosis, identify the causative fungal pathogens and compare the diagnostic validity of fungal fluorescent staining and ITS rDNA PCR-based sequencing with conventional methods including KOH microscopy, mycological culture and histopathology by PAS stain for diagnosis of onychomycosis.
This cross-sectional study was carried out at the Medical Microbiology and Immunology Department, in collaboration with the Central Laboratory, Faculty of Medicine, Menoufia University during the period from January 2020 to April 2021. The study involved 100 patients with different clinical types of onychomycosis (15 males and 85 females with a mean age of 47.36 ±15.204 years old) from those attending the Outpatient Clinic of Dermatology Department, Faculty of Medicine, Menoufia University.
Following patient’s history taking and thorough clinical examination, nail clippings, scrapings or subungual debris from the affected finger and/or toenails were collected in a sterile plastic Petri dish plate or sterile container for processing. Each specimen was evenly divided into five parts. The first portion of specimen was examined directly by 20% KOH mount for the presence of fungal elements in the form of fungal hyphae, pseudohyphae, arthroconidia, spores, or budding cell. The second portion of the nail samples was stained with PAS stain for histopathological examination for the presence of dip magenta-colored hyphae, arthroconidia, or yeasts. The third portion of the nail samples was stained with acridine orange (AO) fluorescent dye and examined using a fluorescence microscope. The fungal fluorescence was identified by tubular or annular elements with a thin rim of bright orange fluorescence outlining the usual shape of the fungal cell wall.
The fourth portion of nail specimen was firstly minced and then divided into two parts to be cultured onto two Sabouraud’s dextrose agar plates (one with cycloheximide and chloramphenicol and the other without). These plates were incubated at 25°C and 37°C for 1-4 weeks and examined daily for colony growth. Dermatophytes were identified macroscopically (color and consistency of the surface and reverse, topography, and texture). For suspected yeast and yeast like growth, microscopic
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examination of isolates was performed after staining by Gram’s stain to evaluate their response to stain, shapes, their arrangement and yeast budding form. A non-dermatophytes mold (NDMs) was only considered to be pathogenic when direct examination was positive and repeatedly isolated on two separate samples, in the absence of any dermatophytes or yeast growth.
The fifth portion of nail specimen was examined by PCR-based internal transcribed spacer (ITS) ribosomal DNA (rDNA) PCR-based sequencing. Fungal DNA was extracted according to the Manufacturer’s instructions. The fungus-specific universal primers ITS1 (5ʹ- TCCGTAGGTGAACCTGCGG-3ʹ) and ITS4 (5ʹ-TCCTCCGCTTATTGATATGC-3ʹ) were used to amplify the full ITS sequence (product size 700-900 bp). PCR was performed for the 100 samples in a thermocycler. PCR products were separated by agarose gel electrophoresis and subsequently subjected to sample purification. Sequencing of 30 purified PCR products was done using Taq DyeDeoxy™ and ABI Prism™ terminator cycle sequencing kits (Applied Biosystems, Foster City, CA, USA) and the above-mentioned PCR primers. The sequencing results were evaluated using the nucleotide Basic Local Alignment Search Tool (BLAST) to determine the closest relatives on the NCBI website (http: //www. ncbi.nlm.nih.gov).
Fifty percent of the studied cases were within the age group of 40-60 years. Female and male patients represented 85% and 15% respectively. Seventy percent of studied patients were housewives while the remaining 30% were manual workers (12%), farmers (9%), laborers (3%) and students (6%). Only15% of patients were smokers. About 62% of the studied patients lived in urban areas and 42%, 32% and 26% belonged to middle, low and high socioeconomic levels respectively.
The most prevalent clinical type of onychomycosis was DLSO (38%) and the mostly affected site was toenails (60%). Disfiguring was the most common symptom (75%), while hyperkeratosis was the most frequent presenting sign (70%). Sixty percent of the patients had a moderate form of onychomycosis and 58% had recurrent onychomycosis. About 45% of patients had acute disease stage while the other 30% and 25% of patients had chronic and sub-acute disease respectively. In all disease stages, there was a female predominance and the most frequent age group was 40-60 years with no statistically significant difference (P>0.05). All clinical types of onychomycosis were more frequent in acute onychomycosis with no significant difference between the three stages of the disease (P>0.05).
About 59%, 46%, 25% and 20% of the studied patients had chronic hepatic diseases, anemia, diabetes and peripheral vascular diseases (PVD) respectively. Notably, 71%, 67% and 54% of the studied patients were excessively exposed to water, chemicals and soil respectively. Regarding immunosuppressed conditions, 56% of the studied cases had malignancy, 68% received chemotherapy, 65% received radiotherapy and 59% received corticosteroid therapy. Only 13% of the studied cases had positive family history, 16% had previous history for nail trauma and only 8% of them used to wear occlusive shoes throughout the year. The highest incidence of onychomycosis was observed in summer (41%).
The most frequent clinical type in toenail and fingernail infection was DLSO representing 43.3 %( 26/60) and 30% (12/40) respectively with no significant statistical difference (P>0.05).
The applied phenotypic diagnostic tests; KOH direct microscopy, PAS staining, fungal culture and fluorescent staining were positive in 52%, 55%, 70% and 80% of the processed nail specimen respectively. There was a statistically significant difference
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between positive and negative results regarding fungal culture and fluorescent staining procedures (P<0.001).
A total of 76 fungal species were isolated from 70 positive fungal cultures (6 cases yielded mixed fungal growth). The highest frequency was for yeast fungi (39/76: 51.3%) of which the most frequently isolated yeast spp. were Candida spp. (28/76: 36.8%). Dermatophytes accounted for 31.6% (24/76) of the totally obtained fungal isolates of which Trichophyton rubrum was the most prevalent species (11/76:14.5%). NDMs represented 17.1 % (13/76) where Aspergillus fumigatus was the predominant species (8/76: 10.5%). Mixed mycotic infections were recovered from 6% of the cultured nail specimens.
Both KOH direct microscopic smear and PAS staining showed no significant statistical difference for detection of dermatophytes, NDM s and yeasts. However, fluorescent staining proved better detection rate for dermatophytes (42.1%).
In the present study, the etiology of onychomycosis proved to be due not only fungal but also bacterial etiology. Mixed fungal and bacterial infection was found in 47.1% (33/70) of onychomycosis cases. The frequently isolated bacterial species associated with dermatophytes and NDMs was S.aureus (93.3% and 72.2% respectively). But, in case of yeast fungi, there was associated S.aureus bacterial infection in 25% of onychomycosis cases.
ITS rDNA PCR-based sequencing was applied for 30 nail specimens for species identification and the results were compared to those of fungal culture. In this study, 98 out of 100 nail specimens (98%) were positive by PCR assay. The results of 30 ITS rDNA PCR-based sequencing were compared with the corresponding 30 fungal cultures results. In 36.7% of samples, yeasts were co-identified by culture and sequencing. Candida spp. detected by culture was established as Candida tropicalis by PCR-based sequencing. Regarding dermatophytes, one Epidermophyton floccosum was previously diagnosed by culture but by PCR-based sequencing, it was diagnosed as Trichophyton mentagrophytes. Regarding NDMs, Aspergillus niger that was previously diagnosed by culture was confirmed as Aspergillus tubingenesis by PCR-based sequencing. Also, one Aspergillus flavus that was previously diagnosed by culture was confirmed as Aspergillus fumigatus by sequencing.
Among toenail infection (n=55), dermatophytes were the most frequently isolated fungi representing 41.8% (23/55), followed by yeasts (36.4%; 20/55) and finally NDMs (21.8%; 12/55) with statistically significant difference (P=0.002), but in case of fingernail infection (n=21), yeasts were the predominantly isolated fungi accounting for 90.4% (19/21) followed by dermatophytes and NDMs which equally represented 4.8% (1/21) with highly significant statistical difference (P<0.001).
In DLSO, TDO and WSO, yeasts were the most frequently isolated fungi accounting for 61.8% (21/34), 46.1% (6/13) and 55.6% (5/9) respectively with no statistical significant difference (P>0.05). On the other hand, in PO, the most frequently isolated fungi were dermatophytes (40%; 24/20) with no statistical significant difference (P>0.05).
Agreement between the diagnostic tests (KOH, PAS, fluorescent staining and PCR) with fungal culture was evaluated by the Cohen kappa test. The agreement of fungal culture with PAS and fluorescent staining was fair (0.396 and 0.315 respectively) which was higher than that with KOH and PCR (slight agreement; 0.024 and 0.091 respectively). Furthermore, a statistically significant difference between PAS,
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fluorescent staining, PCR and culture result was observed (P<0.05). In contrast, no statistically significant difference between KOH mount and culture results (P>0.05) was detected.
Considering fungal culture as the gold standard for diagnosis of onychomycosis, the most sensitive method was PCR (100%) followed by fluorescent staining (89%), PAS staining (69%), and then KOH mount (53 %). Regarding the accuracy of each test, fluorescent staining proved the highest diagnostic accuracy (74%) followed by PCR (72%), PAS staining (71%) and then KOH mount (52%). Concerning the specificity of each test, PAS proved the highest diagnostic specificity (77%) and PCR demonstrated the least specificity (7%).