|Year : 2022 | Volume
| Issue : 1 | Page : 32-36
Efficacy of periodic acid–Schiff stain in the diagnosis of onychomycosis – A cross-sectional study
Mohamed Salahudeen1, Anita Ramdas2, Arthi Elumalai3, Kandasamy Ravichandran4, Sheela Kuruvila1
1 Department of Dermatology, Venereology and Leprosy, Pondicherry Institute of Medical Sciences, Puducherry, India
2 Department of Pathology, Pondicherry Institute of Medical Sciences, Puducherry, India
3 Department of Microbiology, Pondicherry Institute of Medical Sciences, Puducherry, India
4 Department of Biostatistics, Pondicherry Institute of Medical Sciences, Puducherry, India
|Date of Submission||18-Jan-2022|
|Date of Decision||24-Mar-2022|
|Date of Acceptance||25-Mar-2022|
|Date of Web Publication||8-Jul-2022|
Dr. Sheela Kuruvila
Department of Dermatology, Venereology and Leprosy, Pondicherry Institute of Medical Sciences, Puducherry
Source of Support: None, Conflict of Interest: None
Background: Onychomycosis is the fungal infection of the nail which needs to be differentiated from other causes of dystrophic nails due to its long-term treatment. Direct microscopic examination with potassium hydroxide (KOH) mount along with fungal culture shows inconsistent sensitivity. Thus, there is a need of more accurate and efficient method to detect onychomycosis.
Aims and Objectives: This study aims to compare periodic acid–Schiff (PAS) stain with KOH and nail fungal culture in the diagnosis of onychomycosis.
Materials and Methods: This was a cross-sectional study conducted in the dermatology outpatient department of a tertiary care center in South India from July 2019 to July 2021. A total of 93 patients with clinical suspicion of onychomycosis were included in the study and evaluated for the following three methods: KOH mount, nail fungal culture, and PAS stain.
Results: Of the 93 patients, PAS was positive in 82 (88%) cases, KOH mount was positive in 32 (34%) cases, and nail fungal culture showed positive results in 49 (53.2%) patients. The percentage of positivity of combined PAS and culture was 89 (95.7%) and combined KOH and culture was 63 (65.7%). The culture yielded growth in 49 patients showing dermatophytes in 2, yeasts in 35, and nondermatophyte molds (NDMs) in 12 specimens The most common isolated species was Candida tropicalis found in 11 (22.4%) followed by Candida species in 8 (16.3%), Candida albicans in 7 (14.2%), and Trichosporon species in 6 (12.2%) specimens. Among the NDMs, Fusarium oxysporum was the most isolated found in 5 (10.2%).
Conclusion: The importance of this study is to highlight the utility of PAS stain in the diagnosis of onychomycosis which otherwise may be missed with KOH and culture. PAS can be performed as an additional test to KOH mount and nail fungal culture to close the diagnostic gap.
Keywords: Nail fungal culture, onychomycosis, periodic acid–Schiff stain, potassium hydroxide mount
|How to cite this article:|
Salahudeen M, Ramdas A, Elumalai A, Ravichandran K, Kuruvila S. Efficacy of periodic acid–Schiff stain in the diagnosis of onychomycosis – A cross-sectional study. J Curr Res Sci Med 2022;8:32-6
|How to cite this URL:|
Salahudeen M, Ramdas A, Elumalai A, Ravichandran K, Kuruvila S. Efficacy of periodic acid–Schiff stain in the diagnosis of onychomycosis – A cross-sectional study. J Curr Res Sci Med [serial online] 2022 [cited 2023 May 30];8:32-6. Available from: https://www.jcrsmed.org/text.asp?2022/8/1/32/350136
| Introduction|| |
Onychomycosis refers to a fungal infection of the nail. The agents that cause this disease could be dermatophytes, yeasts, and nondermatophyte molds (NDMs). Nail disorders such as lichen planus and psoriasis might show clinical features similar to onychomycosis. Diagnosis of the infection with some laboratory evidence is necessary before treating the patients with systemic antifungal drugs as the treatment duration is long. Direct microscopic examination with potassium hydroxide (KOH) mount along with fungal culture has been considered to be the gold standard for the diagnosis of onychomycosis., However, the yield of fungal culture is still low. Thus, there is a need of more accurate and efficient method to detect onychomycosis. Based on the available literature on the diagnosis of onychomycosis with periodic acid–Schiff stain (PAS) on nail plates, we did a study to assess the sensitivity of PAS stain compared with KOH mount and nail fungal culture.
| Materials and Methods|| |
A prospective, hospital-based, cross-sectional study was conducted on 93 patients with clinical suspicion of onychomycosis attending the outpatient department of a tertiary care teaching hospital for 2 years between July 2019 and July 2021. The study received ethical approval from the institutional ethics committee (IEC: RC/19/60). Informed consent was taken from all patients. A detailed examination of nails was conducted in each of them.
Potassium hydroxide mounts
Nail clippings from the infected nails were taken. A portion of it was immersed in 20% KOH overnight and examined the next day by using light microscopy.
Nail samples were also inoculated in two sets of Sabouraud's dextrose agar (SDA). One set was inoculated in SDA and the other in SDA with antibiotics (chloramphenicol, gentamicin, and cycloheximide). Each set was incubated at 25° C and 37° C for 4 weeks.
Periodic acid–Schiff staining
For PAS staining, nail clippings were fixed in 10% formalin. The specimens were processed and embedded in paraffin blocks. Thin slices of about 3 μm were taken and mounted on glass slides and PAS staining was done. The presence of dark red dots and/or thread-like structures (hyphae) in between the cells of the nail plate was considered positive [Figure 1].
The data were analyzed by IBM SPSS Statistics for Windows (version 20.0 Armonk, New York: IBM Corporation).
Descriptive statistics were presented as numbers and percentages. Cochran's Q-statistic test and McNemar's test with Bonferroni post hoc correction to assess the statistical significant difference between the three tests were done. Sensitivity, specificity, positive predictive value, and negative predictive value were calculated based on different assumptions. P < 0.05 considered statistically significant.
| Results|| |
The study group comprised 93 patients of which 44 were male and 49 were female. The mean age of the patients was 41.7 years with a standard deviation of ± 14.0 and ranged from 18 to 81 years.
The fingernails alone were most frequently involved in 44 (47%) cases followed by the toenails alone in 41 (44%) cases and both in 8 (8%) cases. Distal and lateral subungual onychomycosis was the most common clinical pattern in 72 (77.4%) patients followed by total dystrophic onychomycosis seen in 19 (20.4%) patients. Superficial white onychomycosis was seen in 2 (2.1%) patients.
Of 93 patients, PAS was positive in 82 (88.2%) cases. KOH mount was positive in 38 (40.9%) cases, and mycological culture showed positive results in 49 (52.7%) patients. [Table 1] shows the details of all three tests in the study group. The Cochran's Q-statistic test showed a significant difference between these three tests (Cochran's Q = 41.5, P < 0.001). The McNemar's test with Bonferroni post hoc correction also showed a significant difference (P = 0.001) between PAS and culture as well as PAS and KOH. However, no difference was found between KOH and culture (P = 0.108).
In PAS-positive cases, the fungal elements were seen lying between the laminae of the nail plates and appeared dark pink. The culture yielded growth in 49 patients showing dermatophytes in 2, yeasts in 35, and NDM in 12 specimens [Figure 2] and [Figure 3]. The most common isolated species was Candida tropicalis found in 11 (22.4%) followed by Candida species in 8 (16.3%), Candida albicans in 7 (14.2%), and Trichosporon species in 6 (12.2%). Among the NDM, Fusarium oxysporum was the most isolated found in 5 (10.2%). Dermatophyte was isolated in two patients and both were of species Trichophyton rubrum.
|Figure 2: Species distribution of nondermatophyte molds isolated in culture|
Click here to view
The percentage of positivity of combined PAS and culture was 89 (95.7%) and combined KOH and culture was 63 (67.7%). Comparing PAS test alone as an alternative method of diagnosis the sensitivity was 88.2% [Table 2].
The present study found a sensitivity of 49.0% with KOH mount and 85.7% with PAS staining [Table 3]. The specificity of KOH mount was 68.2% and PAS staining was 9.1%. The positive predictive value of KOH mount was 63.2% while it was 51.2% for PAS staining. The negative predictive value for KOH was 54.5% and for PAS was 36.4%. The sensitivity of PAS is higher when it is calculated considering at least one test as positive compared to considering culture as the gold standard. However, the sensitivity of KOH is higher when culture is considered as the gold standard.
|Table 3: Diagnostic accuracy of potassium hydroxide and periodic acid-Schiff staining|
Click here to view
| Discussion|| |
Onychomycosis is the most common nail disorder and represents about 30% of all superficial mycoses. Three groups of fungi cause this disease: dermatophytes, yeasts, and NDM. The common methods used for the diagnosis of onychomycosis are fungal nail culture and KOH preparation of nail samples. The reported sensitivity of fungal culture for identifying dermatophytes ranges from 25% to 80%, with an estimated 30% false-negative results with KOH and culture studies. Based on the available literature, there are reports on histopathological examination with PAS staining of nail clips which mention it as a highly reliable diagnostic tool for onychomycosis.
In the literature regarding the evaluation of nail histology for diagnosing onychomycosis, PAS was the most commonly used stain., The technique for processing distal nail clipping for the detection of invading fungi was first described by Jilson and Piper in 1957. They have mentioned that the distal part of the nail alone is sufficient in providing histopathological information regarding the existence of mycotic nail infections when compared to the samples obtained from the entire nail. The entire procedure is painless and requires only a few instruments. Suarez et al. have mentioned in their study that nail specimens >3 mm were more likely to be positive for PAS. Individuals with full-thickness onychomycosis may have a low yield on culture from clippings alone because although organisms may be visible with direct microscopy, they may not be viable on culture.
Our results were comparable with the results achieved by other authors who had reported higher sensitivity with PAS as compared to other diagnostic tests [Table 4]. In 30 patients, PAS was the only evidence of fungal infection. If this test had not been done, they would have been falsely reported as negative. Out of 93 patients in our study, 49 patients showed positive fungal culture growth out of which Candida (71%) was the most isolated organism followed by NDM (24%). Only two patients showed growth of T. rubrum. In a study done by Bokhari et al. Candida was the most common fungus isolated. NDM has also been found to be isolated in high numbers in studies conducted by Shenoy et al. and Grover., The prevalence of NDM in onychomycosis is reported in hot and humid tropical and subtropical climates. It is worthwhile to note that both patients with T. rubrum growth in culture did not have any Tinea corporis infection anywhere on the body.
| Conclusion|| |
The importance of this study is to highlight the utility of PAS investigation in diagnosing a case of onychomycosis which otherwise may be missed with KOH and culture. Any delay in the diagnosis of onychomycosis will lead to total nail dystrophy which may be permanent in spite of adequate treatment. PAS can be performed as an additional test to KOH mount and nail fungal culture to close the diagnostic gap.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
| References|| |
Jung MY, Shim JH, Lee JH, Lee JH, Yang JM, Lee DY, et al.
Comparison of diagnostic methods for onychomycosis, and proposal of a diagnostic algorithm. Clin Exp Dermatol 2015;40:479-84.
Bertanha L, Chiacchio ND. Nail clipping in onychomycosis. An Bras Dermatol 2016;91:688-90.
Yue X, Li Q, Wang H, Sun Y, Wang A, Zhang Q, et al.
Scanning electron microscopy of the nail plate in onychomycosis patients with negative fungal culture. Scanning 2016;38:172-6.
Haneke E. Fungal infections of the nail. Semin Dermatol 1991;10:41-53.
Suarez SM, Silvers DN, Scher RK, Pearlstein HH, Auerbach R. Histologic evaluation of nail clippings for diagnosing onychomycosis. Arch Dermatol 1991;127:1517-9.
Weinberg JM, Koestenblatt EK, Tutrone WD, Tishler HR, Najarian L. Comparison of diagnostic methods in the evaluation of onychomycosis. J Am Acad Dermatol 2003;49:193-7.
Lui HN, Lee DD, Wong CK. KONCPA: A new method for diagnosing Tinea ungium
. Dermatology 1993;187:166-8.
Mehregan DA, Mehregan DR, Rinker A. Onychomycosis. Cutis 1997;59:247-8.
Jilson OF, Piper EL. The localization of fungi within human nails. J Invest Dermatol 1957;28:137-46.
Davies RR. Mycological tests and onychomycosis. J Clin Pathol 1968;21:729-30.
Shenoy MM, Teerthanath S, Karnaker VK, Girisha BS, Krishna Prasad MS, Pinto J. Comparison of potassium hydroxide mount and mycological culture with histopathologic examination using periodic acid-Schiff staining of the nail clippings in the diagnosis of onychomycosis. Indian J Dermatol Venereol Leprol 2008;74:226-9.
] [Full text]
Wilsmann-Theis D, Sareika F, Bieber T, Schmid-Wendtner MH, Wenzel J. New reasons for histopathological nail-clipping examination in the diagnosis of onychomycosis. J Eur Acad Dermatol Venereol 2011;25:235-7.
Jeelani S, Ahmed QM, Lanker AM, Hassan I, Jeelani N, Fazili T. Histopathological examination of nail clippings using PAS staining (HPE-PAS): Gold standard in diagnosis of onychomycosis. Mycoses 2015;58:27-32.
Blake N, Zhu J, Hernandez G, Juliano PJ. A retrospective review of diagnostic testing for onychomycosis of the foot. J Am Podiatr Med Assoc 2015;105:503-8.
Agha H, Noreen F, Raza N, Malik NA. Comparison of yields of KOH mount and PAS staining of nail clippings for hyphae in cases of onychomycosis. Pak Armed Forces Med J 2018;68:80-4.
Lin YC, Sun PL, Hsiao PF, Sun FJ, Wu YH. Methods for diagnosing onychomycosis: A comparative study of 459 cases. Dermatol Sin 2019;37:63-6. [Full text]
Begari V, Pathakumari P, Takalkar AA. Comparative evaluation of KOH mount, fungal culture and PAS staining in onychomycosis. Int J Res Dermatol 2019;5:554-8.
Gautam M, Shah N, Bhattar P, Nadkarni N, Patil S. Comparative evaluation of potassium hydroxide mount, fungal culture, and histopathology of nail clipping with periodic acid-Schiff stain in the diagnosis of onychomycosis. Indian J Dermatopathol Diagn Dermatol 2021;8:6-12. [Full text]
Bokhari MA, Hussain I, Jahangir M, Haroon TS, Aman S, Khurshid K. Onychomycosis in Lahore, Pakistan. Int J Dermatol 1999;38:591-5.
Grover S. Clinico-mycological evaluation of onychomycosis at Bangalore and Jorhat. Indian J Dermatol Venereol Leprol 2003;69:284-6.
] [Full text]
Tosti A, Piraccini BM, Lorenzi S. Onychomycosis caused by nondermatophytic molds: Clinical features and response to treatment of 59 cases. J Am Acad Dermatol 2000;42:217-24.
[Figure 1], [Figure 2], [Figure 3]
[Table 1], [Table 2], [Table 3], [Table 4]