Nail Clipping with PAS Stain and Correlation with Fungi Isolated in Culture: A Valuable Exchange in the Diagnosis of Onychomycosis

Abstract

Introduction

The etiological agents of onychomycosis are dermatophyte fungi, nondermatophyte filamentous fungi (NDFF), and yeasts. Nail clippings are an accurate tool for diagnosing onychomycosis. The objective of this study was to correlate the characteristics of fungi observed in nail clipping examinations with the results for agents isolated in culture, indicating whether the fungus found on microscopy is a dermatophyte, NDFF, or yeast.

Methods

An observational descriptive study of onychomycosis nail clippings stained with PAS comparing culture results with microscopic criteria of fungi morphology, arrangement/orientation, and location of organisms in the nail and presence of neutrophils, serous lakes, and bacterial colonies was conducted.

Results

Thirty-three nail clippings from 29 participants were analyzed. A statistically significant correlation (p < 0.05) was found between smooth/thin/long hyphae with a “combed” pattern and dermatophyte agents (sensitivity 89.4%, specificity 91.5%), and between irregular/thick/short anarchic distributed hyphae and nondermatophytes (sensitivity 61.5%, specificity 89.4%). No statistical association with region where fungi were in the nail or with the presence of neutrophils, serous lakes or bacteria was found. Small, rounded, yeast-like organisms correlated with Candida infection.

Conclusions

Although not a substitute for culture, microscopic analysis of fungal structures offers good accuracy for differentiating agents of onychomycosis.

Introduction

The main etiological agents of onychomycosis are dermatophyte fungi, predominantly Trichophyton rubrum (in up to 70% of cases) and Trichophyton mentagrophytes (up to 20%). Other possible agents include nondermatophyte filamentous fungi (NDFF), such as the species Fusarium, Neoscytalidium, Scopulariopsis, and Aspergillus (10% of cases) and, more rarely, yeasts [1, 2]. Recent studies have shown an increase in the proportion of NDFF and yeast, with each agent accounting for up to 20% of cases isolated, a shift which has direct implications for the therapeutic approach for this infection [3, 4].

The most used tools for diagnostic confirmation are direct mycological examination, culture for fungi and nail clippings [5]. Culture for fungi, although considered the gold-standard in diagnosis, is associated with high rates of false-negatives (as high as 40%) and has the drawback of taking longer to obtain results. Concomitant use of the 3 methods significantly improves the sensitivity of the diagnosis [6].

The primary objective of this study was to correlate the characteristics of fungi observed on nail clipping examinations with results for agents isolated in culture. These correlations may provide an indicator, in some cases, as to whether the fungus found in nail clippings is a dermatophyte, NDFF or yeast.

Materials and Methods

A descriptive observational study of nail clippings collected from patients clinically diagnosed with onychomycosis and positive for fungi on culture was carried out [3]. Patients with clinical suspicion of onychomycosis provided nail clipping samples for microscopic analysis and subungual scrapings to perform culture for fungi. None of the patients had received topical or systemic anti-fungal treatment in the 3 months leading up to sample collection. The nail fragments for microscopic examination were collected using nail clippers and placed in a vial containing 10% formalin solution. The processing technique was performed as described by Werner and Antunes [7], and slides were stained using the Periodic Acid-Schiff (PAS) method with digestion. For cultures, seeding was performed in 2 tubes containing Sabouraud dextrose agar (Difco, USA) and 2 containing Mycosel® (Difco, Franklin Lakes, NJ, USA). The agents were considered as NDFF after 3 collections performed at least 1 week apart, showing exclusive growth of the same fungus and microculture in potato agar with subsequent microscopic morphological analysis. Cases not exhibiting fungi isolation on cultures, or whose sample material proved inadequate for clipping analysis, were excluded from the study.

The microscopic analysis 5–10 slides per sample of the clippings was carried out by one of the authors (B.W., dermopathologist), blinded to the culture results. Few variations in criteria grading within the same sample's slides were seen and, when present, the mean of all slides was considered. The following morphological criteria of the fungi were recorded: presence of smooth/thin/long hyphae, presence of irregular/thick/short hyphae, presence of branching, presence of arthroconidia or arthrospores, yeast-like forms, and arrangement/orientation of the organisms in the nail (whether oriented in a particular direction conferring a “combed” pattern or arranged in an anarchic fashion). Additional microscopic parameters analyzed were location of organisms in the nail (plate or subungual region), and the presence or absence of neutrophils, serous lakes and bacterial colonies. The denomination of long or short hyphae was given by the proportion between the diameter and length of the hypha, being considered long when the length was greater than or equal to three times its diameter, and short if less than three times. When smooth/thin/long and irregular/thick/short hyphae were seen in equal proportions in slides, these data were counted in both categories.

After completion of the microscopic evaluation, the codes were revealed for the respective results of the fungal cultures. Photomicrographs were done using the ×40 objective to record the key morphological features of the fungi observed. In cases where image enlargement was required to better visualize details, care was taken to apply standardized parameters to all photomicrographs, replicating the same scale.

Statistical analysis of the parameter data was performed using the χ² test and test for comparing means between dermatophyte and NDFF for each criterion and between filamentous and Candida sp. for yeast-like forms. Diagnostic test evaluation with sensitivity and specificity was calculated separately for smooth/thin/long, irregular/thick/short hyphae, “combed,” and anarchic pattern, carried out based on contingency tables only with the 32 samples of filamentous fungi.

Results

A total of 52 samples from 39 patients clinically diagnosed with onychomycosis were included in the study. Of these samples, 12 were excluded for absence of fungal growth and 7 for absence of subungual region in the clipping specimen. Thus, a final total of 33 nail clippings from 29 participants were analyzed. Out of the cases analyzed, 29 samples were taken from toenails, particularly the 1st toe. Four samples were from the fingernails.

Among the cultures, 22 cases involved infection by dermatophytes (66.67%), where 20 had growth of T. rubrum and 2 of T. mentagrophytes. Of the 10 cases of NDFF (30.30%), 6 were Fusarium sp., 3 Neoscytalidium hyalinum, and 1 involved Neoscytalidium dimidiatum. Candida sp. growth was detected in 3 cases, comprising 1 of yeast alone (3.03%) and 2 of the yeast plus Fusarium sp. Those 2 cases with Fusarium sp. were considered only as NDFF.

Most of the fungi were found in the subungual region, accounting for 30 cases (90.90%). For the nail plate, fungi were found in 19 samples (57.58%). The presence of fungi in both the subungual region and the nail plate occurred in 16 cases (48.48%). Dermatophytes were found in the subungual region in 90.91% of cases and NDFF in 90.00%, although this difference was not statistically significant (p = 0.0710).

In 22 cases, smooth/thin/long hyphae were observed, 19 of which were associated with dermatophyte fungal cultures (shown in Fig. 1). Irregular/thick/short filamentous organisms overlapping and arranged anarchically were observed in 19 cases, 10 of which involved infection by NDFF (shown in Fig. 2) and 9 by dermatophyte fungi (shown in Fig. 3). In 6 cases, regular rounded yeast structures were present, identified as Candida growth in culture in 3 cases. Two of these cases involved growth of both Candida sp. and Fusarium sp., while one case involved growth of Candida sp. alone (shown in Fig. 4). Comparison of the morphology of the fungal structures against culture results revealed a statistically significant relationship between smooth/thin/long hyphae and dermatophyte agents (p = 0.0017) and irregular/thick/short hyphae with NDFF (p = 0.0019) (shown in Table 1). All 5 clipping specimens that exhibited arthroconidia (shown in Fig. 4) had dermatophyte fungal growth in culture, although this correlation was not statistically significant (p = 0.1062). Branching was observed in both dermatophyte fungi and NDFF (5 cases each), albeit slightly more prevalent in NDFF, without reaching statistical significance.

Fig. 1.

Photomicrographs of 6 cases of onychomycosis infected by filamentous dermatophyte fungi (T. rubrum) showing smooth/thin/long hyphae with tendency to be arranged in a parallel fashion. This arrangement of the fungi confers a “combed” appearance. Periodic acid-Schiff, with digestion − objective ×40.

Fig. 2.

Photomicrographs of 6 cases of onychomycosis infected by filamentous dermatophyte fungi (T. rubrum) showing major morphological variation in the appearance of organisms. Arthrospore formation, thick hyphae and prominent septation, or irregular rounded structures are evident. Periodic acid-Schiff, with digestion − objective ×40.

Fig. 3.

Photomicrographs of onychomycosis involving infection by NDFF. a–cNeoscytalidium dimidiatum with large organisms often with bizarre appearance or thick hyphae resembling tree branches. d–fNeoscytalidium hyalinum with thick hyphae and bifurcations or branching. g–iFusarium sp. With thin, sometimes branched, hyphae, or thick irregular hyphae arranged erratically. Periodic acid-Schiff, with digestion − objective ×40.

Fig. 4.

Photomicrographs of nails infected by Candida sp. alone (left-hand side figure), coinfected with Fusarium sp. (center figure) and coinfected with T. rubrum (right-hand side figure). Candida sp. yeasts are rounded and regular, in contrast with the irregular Fusarium sp. and T. rubrum hyphae. Periodic acid-Schiff, with digestion − objective ×40.

Table 1.

Statistical comparison of morphological criteria and distribution of fungi

Criteria evaluated	Total	Dermatophytes, n (%)	NDFF, n (%)	Candida, n (%)	95% CI	p value
Culture results	33	22 (66.67)	10 (30.30)	1 (3.03)‡	−	−
Morphological
Presence of smooth/thin/long hyphae	22	19 (86.36)	3 (30.00)	−	20.20–77.54	0.0017
Presence of irregular/thick/short hyphae	19	9 (40.91)	10 (100.00)	−	24.67–76.74	0.0019
Presence of branching hyphae	10	5 (22.73)	5 (50.00)	−	(−) 6.24–56.47	0.1289
Presence of arthroconidia	5	5 (22.72)	0 (0.00)	−	(−) 7.75–43.44	0.1062
Yeast-like structures*	6	5 (15.63)		1 (100.00)	3.40–93.13	0.0339
Filamentous± X Candida
Arrangement of hyphae
Combed†	19	17 (77.27)	2 (20.00)	−	20.01–76.36	0.0026
Anarchic	13	5 (22.73)	8 (80.00)	−	20.01–73.36	0.0026

CI, confidence interval, NDFF; nondermatophyte filamentous fungi.

^*Regular rounded structures.

^±All dermatophyte and nondermatophyte filamentous infections, as opposed to Candida sp. cases.

^†Well-organized hyphae, resembling bundles in different directions.

^‡One case of Candida sp. growth alone, but 2 further cases of coinfection by Candida sp. plus Fusarium sp. considered only as NDFF.

The “combed” pattern of hyphae in nail clippings was evident in 17 dermatophyte samples and completely absent in five samples. This same pattern was seen in 2 NDFF cases and totally absent in 8 cases, representing a statistically significant association of the “combed” pattern with dermatophyte agents and of the anarchic pattern with nondermatophyte agents (p = 0.0026) (shown in Table 1).

Five cases (15%) contained few fungi or the sample was small, owing to the low number of nail fragments available for examination or to the small amount of the subungual region sampled (shown in Fig. 5). Neutrophils were found in 8 clippings (24.24%), serous lakes in 21 (63.64%), and bacteria in 22 (66.67%) (shown in Table 1). No statistical difference was found for the presence of these features in the culture results for dermatophyte fungi, NDFF, or Candida (p > 0.10).

Fig. 5.

Photomicrographs of nails with fungal infection with single growth in the sample examined containing few organisms. In these cases of culture results for T. rubrum, T. mentagrophytes, and Fusarium sp., respectively, differentiation between dermatophyte and nondermatophyte filamentous fungi in nail clippings proves difficult. Periodic acid-Schiff, with digestion − objective ×40.

Discussion

The assessment determining the distribution of the fungi in the nail showed that the most affected site was the subungual region (90.91% of cases). Although fungi location proved nonsignificant for differentiating between dermatophytes and NDFF, it is paramount that the sample contains this part of the nail to allow a thorough microscopic analysis and serves as a warning against collecting subungual scrapings for fungal culture immediately before carrying out the nail clipping procedure.

Of the 33 cultures, 22 detected dermatophyte fungi. The main microscopic findings in nail clippings which are statistically more typical in this fungal class are regular hyphae (generally rounded, smooth/thin/long − sensitivity 86.36%, specificity 70.0%, and accuracy 81.25%) and the well-organized arrangement of agents in bundles conferring a “combed” appearance (sensitivity 89.47%, specificity 61.54%, and accuracy 78.12%).

Comparative dermatological analysis has produced important findings in infectious diseases in demonstrating that patterns of clinical and dermatoscopic parasitism repeat on histology and confocal microscopy [8, 9, 10]. A recent study showed that nail dermoscopy (onychoscopy) in onychomycosis reveals a statistically significant pattern of yellowed-longitudinal lines or striations (spiked or “fringe” pattern) for the diagnosis of nail fungus infection [11]. This aspect is caused by penetration of filamentous fungi into the nail plate at the hyponychium with longitudinal spread in the direction of nail growth. The “combed” aspect of the dermatophyte fungi on microscopy reflects the spiked pattern of onychoscopy. In fact, the “spiked” and “combed” aspects are visualizations of the same fungal arrangement but are visualized using examinations with different enlargements.

Arthroconidia or arthrospores are structures of resistance formed by breakage or disarticulation of fungal mycelium (arthrosporic phenomenon), and characteristic features of dermatophytes [4]. The present study is the first to report the presence of these structures on microscopy examination of nail clippings. Arthroconidia have yeast-like morphology but are more polygonal and are often arranged in chains (catenulate), showing the silhouette of the hitherto complete hyphae. The nuclei are sometimes visible and can be differentiated from true yeasts since the latter are more rounded and arranged in clusters, as opposed to a catenulate fashion. Arthroconidia were found in clippings of 5 cases, all of which were infected by dermatophyte fungi. Although this study was not statistically significant on comparison with NDFF (p = 0.1062), this finding on direct mycological examination is pathognomonic of infection by dermatophyte fungi [4].

In the 10 cases exhibiting NDFF growth, there were irregular/thick/short filamentous organisms (sensitivity 52.63% and accuracy 71.88%) overlaid in an anarchic arrangement. However, dermatophyte fungi also displayed irregular shapes, albeit rarer than the long, thick smooth hyphae typically found. It is unclear why marked morphological changes occur in dermatophytes, but fungal viability is likely influenced by the interaction with the immune response of the host [12]. The presence of yeast-like structures proved statistically significant for differentiating between filamentous fungi and yeasts (p = 0.0339).

For 2 cases, microscopic analysis detected the presence of both hyphae and yeasts concomitantly. On cultures, growth of Fusarium sp. and Candida sp. was found in one case, and T. rubrum in the other. This finding illustrates the fact that the nail microenvironment contains contaminants, such as yeasts and NDFF, alongside dermatophyte pathogens. Isolation of these contaminants in cultures should be assessed using criteria [3, 4].

The criteria of presence of neutrophils, serous lakes, and bacteria did not differ statistically for percentage of cases infected by dermatophytes, NDFF, or Candida sp. Neutrophils were found in 24.24% of cases, a rate higher than that seen in ungual psoriasis [13, 14]. Therefore, if neutrophils are detected in nail clippings, it is vital to rule out fungal infection before establishing a diagnosis of psoriasis.

Statement of Ethics

This study protocol was reviewed and approved by CEP (Comitê de Ética em Pesquisa − Research Ethics Committee) 9587 − Secretaria de Saúde de São José dos Pinhais (#51533521.4.0000.9587). Written informed consent was obtained from participants who participate in the study.

Conflict of Interest Statement

The authors have no conflicts of interest to declare.

Funding Sources

There were no sources of funding for this work.

Author Contributions

Betina Werner: collection, analysis, interpretation of data, and reviewing of the report. Flávia Trevisan: analysis, interpretation of data, and writing of the report. John Verrinder Veasey: collection, analysis, interpretation of data, and reviewing of the report.

Data Availability Statement

All data generated or analyzed during this study are included in this article. Further enquiries can be directed to the corresponding author.