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 Table of Contents  
ORIGINAL ARTICLE
Year : 2021  |  Volume : 8  |  Issue : 1  |  Page : 6-12

Comparative evaluation of potassium hydroxide mount, fungal culture, and histopathology of nail clipping with periodic acid–Schiff stain in the diagnosis of onychomycosis


1 Department of Dermatology, Venereology and Leprosy, Dr. D.Y. Patil Hospital, Navi Mumbai, Maharashtra, India
2 Department of Dermatology, Dr. R. N. Cooper Hospital, Mumbai, Maharashtra, India
3 Dr. Prachi Bhattar’s Skin, Hair Clinic & Cosmetology Centre, Raipur, Chhattisgarh, India

Date of Submission09-Sep-2020
Date of Decision20-Feb-2021
Date of Acceptance09-Jun-2021
Date of Web Publication11-Aug-2021

Correspondence Address:
Nidhi Shah
Department of Dermatology, HBT Medical College and Dr. R.N. Cooper Hospital, Juhu, Mumbai, Maharashtra.
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/ijdpdd.ijdpdd_94_20

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  Abstract 

Context: Onychomycosis (OM) is a commonly encountered problem in clinical practice, which needs to be differentiated from other causes of dystrophic nails as its treatment is long-term and may have potential side effects. Routinely used laboratory tests like direct microscopy with potassium hydroxide (KOH) and fungal culture (FC) show inconsistent sensitivity. Hence, newer methods of diagnosis are required. Aim: This study aims to compare the efficacy of KOH mount, fungal culture, and histopathology of nail clippings with periodic acid–Schiff stain for the diagnosis of onychomycosis. Settings and Design: This was a cross-sectional study conducted in the Dermatology Outpatient Department of a tertiary care center in Navi Mumbai from September 2013 to September 2014. Subjects and Methods: A total of 102 patients with clinically suspected OM were included in the study and evaluated for the following three methods: KOH mount, FC, and HP/PAS stain. To determine the efficacy and performance characteristics of each test, FC was chosen as the gold standard for statistical analysis. Statistical Analysis Used: Sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) were calculated for each test. Association among the study group was assessed with the help of Chi-square test. Results: Of the 102 patients, direct microscopy with KOH mount was positive in 47, FC in 30, and HP/PAS in 71 patients. Ninety-one out of the 102 patients had at least one of the three diagnostic tests positive. Using this as the denominator, KOH mount, culture, and HP/PAS had sensitivities of 51.64%, 32.96%, and 78.02%, respectively. Diagnostic sensitivity increased to 89.38% when both KOH and HP/PAS were combined.For calculation of specificity of KOH and HP/PAS, we used FC as the gold standard because it gives precise identification of the pathogen. Specificity for KOH and HP/PAS was 59.7% and 34.7%, respectively. It increased to 73.7% if both KOH and HP/PAS were positive. Conclusion: Using PAS on nail scraping can yield higher diagnostic sensitivity with no loss of specificity and hence can be considered as the method of choice for the diagnosis of OM. Accuracy of diagnosis can be increased by combining the two tests (KOH and HP/PAS) instead of one test (HP/PAS).

Keywords: Fungal culture, onychomycosis, periodic acid-Schiff staining, periodic acid-Schiff, potassium hydroxide mount, potassium hydroxide


How to cite this article:
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

How to cite this URL:
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 [serial online] 2021 [cited 2021 Dec 2];8:6-12. Available from: https://www.ijdpdd.com/text.asp?2021/8/1/6/323707




  Introduction Top


Onychomycosis (OM) is a fungal infection of the nails that can be caused by dermatophytes, yeasts, or nondermatophyte molds (NDM). It needs to be differentiated from nail disorders such as psoriasis, lichen planus, eczema, and nail trauma. A correct diagnosis of OM using a laboratory test is essential as the treatment is long term and may have potential side effects.

Various techniques such as direct microscopy with potassium hydroxide mount (KOH) and fungal culture (FC) are commonly used for the laboratory diagnosis of OM. However, KOH preparation is regarded as having a low sensitivity and FC takes a longer time.

Histopathological examination of nail clipping with periodic acid–Schiff stain (HP/PAS) is emerging as a simple, time-efficient test with higher sensitivity. It is also recommended in cases with strong clinical suspicion but shows negative results with culture and KOH.

This study was conducted to compare the efficacy of KOH mount, FC, and HP/PAS stain for diagnosis of OM.


  Subjects and Methods Top


This was a cross-sectional study conducted in the Dermatology Outpatient Department of a tertiary care center in Navi Mumbai from September 2013 to September 2014.

A total of 102 patients (57 males, 45 females) aged 11–78 years (mean: 40.63 years) with clinically suspected OM were included in the study. A case of OM was defined as clinical morphology plus at least one positive test result which is generally accepted in clinical practice. Clinically, the disease was classified as distal and lateral subungual OM, proximal subungual OM (PSO), white superficial OM (WSO), candidal OM (CO), endonyx OM, and total dystrophic OM (TDO).

All clinical variants of OM were included and written informed consent was obtained. However, patients who had taken treatment in the form of systemic and/or topical antifungals in the last 4 weeks were excluded.

Institutional Ethics Committee permission was obtained prior to the start of the study.

Specimen collection

Affected nails were first cleaned with normal saline to remove contaminants and clipped short using standard nail clippers.

For distal and PSO (DLSO and PSO), the abnormal nail was clipped proximally as close to the cuticle as possible and the nail bed and undersurface of the nail plate were scraped with a No.15 surgical blade.

For superficial white onychomycosis (SWO), the white spots on the nail were scraped and the superficial material was discarded; the white debris directly underneath was then collected.

The collected specimen was divided into three parts:

  • Part 1 – Direct microscopy with 10% potassium hydroxide preparation


  • Part 2 – Culture


  • Part 3 – Histopathology with PAS stain


  • Potassium hydroxide preparation

    Standard procedure was followed using 10% KOH and incubation was done for 24 h. The test was considered positive if hyphae and/or spores were detected under the microscope [Figure 1].
    Figure 1: Potassium hydroxide mount showing thin septate hyphae and spores suggestive of onychomycosis under a light microscope

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    Fungal culture

    After inoculation of sample on Sabouraud Dextrose agar (HiMedia Laboratories) with and without antibiotics (0.040g/l chloramphenicol and 0.500g/l cycloheximide), cultures were examined at weekly intervals for growth over 6 weeks. If growth was present, species identification was done based on colony morphology and microscopic appearance [Figure 2][Figure 3][Figure 4][Figure 5][Figure 6].
    Figure 2: Sabouraud’s dextrose agar tube showing slightly raised, white to cream, suede-like colonies with a red reverse suggestive of Trichophyton rubrum

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    Figure 3: Lactophenol cotton blue mount showing microconidia along sides of hyphae suggestive of Trichophyton rubrum (×10)

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    Figure 4: Sabouraud’s dextrose agar tube showing a dense layer of dark brown to black Aspergillus Niger colonies

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    Figure 5: Lactophenol cotton blue mount showing conidia covering the entire surface of the vesicle suggestive of Aspergillus niger (×10)

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    Figure 6: Sabouraud’s dextrose agar tube showing creamy white Candida colonies

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    HP/Periodic Acid–Schiff staining

    Nail specimens for histopathology were obtained with the use of standard nail clippers. The distal free edge of the nail plate, along with any attached subungual material, was clipped just distal to its attachment to the nail bed. The clippings were then fixed in 10% formalin solution.

    Before processing, the nail clippings were placed in 20% KOH to soften the nail for 2–4 h; it was then dehydrated, embedded in paraffin for 6 h, cut with a microtome into thin slices of 4 μm, and stained afterward with PAS stain.

    Demonstration of deeply stained red dots (spores) [Figure 7] and/or thread-like structures (hyphae) [Figure 8] was considered positive for PAS stain.
    Figure 7: Periodic acid–Schiff stain (×40) showing fungal spores

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    Figure 8: Periodic acid–positive intensely stained reddish thread-like fungal hyphae within the nail (×40)

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    To determine the efficacy and performance characteristics of each test, FC was chosen as the gold standard for statistical analysis.

    The following were calculated for each test: sensitivity, specificity, PPV, and NPV.

    Association among the study group was assessed with the help of Chi-square test.


      Results Top


    Of the 102 patients, 57 were male and 45 were female. Their ages ranged from 11 to 78 years with a mean age of 40.6 years.

    Nail involvement and clinical pattern of OM are shown in [Table 1] and [Table 2], respectively.
    Table 1: Nail involvement

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    Table 2: Clinical pattern of onychomycosis in the study group

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    On investigating these patients, 91 out of the 102 patients had at least 1 of the 3 diagnostic tests positive [Table 3].
    Table 3: Summary of test results

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    Direct microscopy with KOH mount was positive in 47 patients (46.08%).

    Culture yielded growth in 30 patients (29.4%), with dermatophytes in 18 (60%), Candida in 3 (10%), and NDM being the causative agent in 9 (30%) patients [Table 4].
    Table 4: Fungus isolated on culture

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    NDM can be laboratory contaminants and can occasionally cause OM. Hence if they are isolated, they are considered positive only if KOH or HP/PAS demonstrates atypical hyphae associated with NDM or if the same organism is repeatedly isolated.[1] In our study, 9 out of a total of 11 NDMs that were isolated qualified as true pathogens after application of these criteria. Trichophyton rubrum was the most common organism isolated.

    In 71 patients (69.6%), HP/PAS demonstrated hyphae and/or spores.

    Thirteen patients showed only KOH positivity, four patients showed only FC positivity, and in 32 patients, HP/PAS was the only evidence of fungal infection. Thus, PAS staining gave a higher yield compared to other tests. Out of all the three diagnostic tests, FC was the least sensitive (32.96%) and HP/PAS (78.02%) was the most sensitive test [Table 5]. Diagnostic sensitivity was increased to 89.38% when the two tests (KOH and HP/PAS) were combined.
    Table 5: Positive results and sensitivity

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    According to the clinical pattern of OM, tests were compared and the results are shown in [Table 6].
    Table 6: Comparison of tests with a clinical pattern of onychomycosis

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    For calculation of specificity of KOH and HP/PAS, we used FC as the gold standard because it gives precise identification of the pathogen. Specificity for KOH and HP/PAS was 59.7% and 34.7%, respectively. It increased to 73.7% if both KOH and HP/PAS were positive.

    HP/PAS had higher sensitivity than KOH, while KOH had higher specificity than HP/PAS, but the difference in both was not statistically significant. (P = 0.069 and 0.2,5 respectively, Pearson’s Chi-square test).

    PPV was highest with KOH mount (38.30%).

    NPV of the both tests was almost equal with KOH mount (78.18%) and PAS staining (78.13%). The results are listed in [Table 7].
    Table 7: Comparison of different tests with culture as the gold standard

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      Discussion Top


    OM is a fungal infection of nails caused by dermatophytes, yeast, or NDM. Increasingly, OM is being viewed as more than a mere cosmetic problem. In spite of improved personal hygiene and living environment, OM continues to spread and persist.

    In our study, the highest incidence (43.13%) was seen between the age group 21–40 years with a mean age of 40.63 years. The similar incidence in 21–40 years age group has been observed in studies done by Jesudanam et al. (59.8%)[2] and Sen et al. (79.4%).[3] The highest incidence in this age group is possibly due to occupation-related trauma, increased exposure to wet work, and cosmetic concerns of younger patients.

    Our study showed that the incidence among males was slightly higher than that of the females with a male-to-female ratio being 1.3:1. Garg et al. (3:1),[4] Sen et al. (1.6:1),[3] and Grover (1.6:1)[5] also observed higher incidence in males in their respective studies. However, Jesudanam et al.[2] reported a slightly higher incidence among females (1.08:1). In females, incidence can be attributed to the higher exposure to water during household work, while in men, it can be attributed to the use of closed footwear.

    In our study, the most common pattern of OM observed was DLSO (53%), followed by TDO (16%), PO (10.78%), MO (8.82%), CO (7.84%), and SWO (4.9%). These findings are in accordance with reports by Garg et al.,[4] Grover[5] and Sen et al.[3] In contrast, Jesudanam et al.[2] reported CO as the most common type in their study.

    Routine laboratory tests like KOH or FC have been employed to confirm the diagnosis in suspected cases of OM. However, only 10%–40% of cases on KOH examination and 10%–30% of cases on culture give a positive yield.[6] Considering the variable sensitivities of KOH and prolonged waiting period for culture, more reliable tests are required for an accurate and prompt diagnosis of OM.

    In recent years, histopathologic examination with PAS stain (HP/PAS) of nail clippings has emerged as a useful test which detects the presence of certain polysaccharides in the walls of hyphae.

    The quality of sample collection and transportation are very important for accurate laboratory diagnosis of OM. Detection of fungal elements by different laboratory tests depends on the site from where the sample is collected. In tropical areas, where molds are more likely to cause OM, samples should include nail plates, nail beds, and subungual debris. The sample should be taken from the proximal and lateral edges of the nails when Candida is suspected. It is very important to take aseptic precautions so that the sample is not contaminated as it can suppress the growth of pathogenic fungi in culture. Adequate samples should be collected to get a higher yield of fungal elements, thereby increasing the chances of obtaining positive results. Inadequate samples can lead to false-negative results due to scanty pathogens.[7]

    Our study compared the following three diagnostic tests – KOH mount, FC, and HP/PAS which can be used in an outpatient setup.

    Out of the 102 patients in our study, 47 (46.08%) were positive for KOH mount, 30 (29.41%) showed culture positivity, and 71 (69.6%) were positive on PAS stain. Out of the 30 patients who showed FC positivity, dermatophytes were isolated in 18 (60%), followed by nondermatophyte in 9 (30%) and Candida in 3 (10%) cases.

    Of the dermatophytes, T. rubrum (23.07%) was the most common organism isolated. The high prevalence of T. rubrum has been explained by its better adaptation to the hard keratin of nails. Similar results have been reported by Sen et al.[3]

    In contrast, Bokhari et al.[8] and Rigopoulos et al.[9] reported Candida as the most common fungus isolated in their studies. NDMs were isolated in 22 of the total 35 patients in the study conducted by Rigopoulos et al.[10]

    Comparing the sensitivity of the different tests, we found PAS staining to be the most sensitive test for the diagnosis of OM in our study.

    Our results were comparable with the results achieved by other authors such as Weinberg et al.[6] and Shenoy et al.[10] who also reported higher sensitivity with PAS as compared to other diagnostic tests [Table 8].
    Table 8: Comparison of sensitivity of tests with other studies

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    In Weinberg et al. study,[6] 105 patients with OM were evaluated using the following diagnostic tests – KOH mount, FC, HP/PAS, and calcofluor white stain. With calcofluor white stain as the gold standard, KOH mount, FC, and HP/PAS had sensitivities of 80%, 59%, and 92%, respectively.

    Similarly, Shenoy et al.[10] described KOH positivity in 50%, FC positivity in 36%, and PAS positivity in 76% of cases.

    In a study done by Wilsmann-Theis et al.,[11] PAS was found to be the most sensitive single test for the diagnosis for OM with a sensitivity of 82%, followed by culture (53%) and direct microscopy (48%). In contrast, Alkhayat et al.[17] and Machler et al.[18] reported lower PAS positivity of 38% and 33%, respectively. We feel that these varying rates could be because of different methods of sampling the nail as well as variations in the method of staining.

    In our study, the diagnostic sensitivity increased to 89.38% when both KOH and HP/PAS were combined in comparison to the sensitivity when only HP/PAS was done (sensitivity: 78.02%, n = 71). When all three tests were performed, 91 out of 102 patients were found to be positive for at least one test. However, FC usually requires weeks to identify fungal species and has the lowest positivity rate (value). There were only three patients where FC came positive and the other two tests were negative. Hence, our results show that the diagnostic accuracy improves dramatically when two tests (KOH and HP/PAS) are combined as against HP/PAS alone. Addition of a third test only marginally increases the diagnostic accuracy. Hence, we recommend a combination of KOH with HP/PAS for a more accurate diagnosis of OM instead of HP/PAS alone.

    For calculation of specificity of KOH and HP/PAS, we used FC as the gold standard because it gives precise identification of the pathogen.

    HP/PAS had higher sensitivity than KOH, while KOH had higher specificity than HP/PAS, but the difference in both was not statistically significant (P = 0.069 and 0.25, respectively, Pearson’s Chi-square test).

    PPV was highest with KOH mount (38.30%).

    NPV of the both tests was almost equal with KOH mount (78.18%) and PAS staining (78.13%).

    The specificity of KOH was more than PAS in our study which is in accordance with other studies such as Blake et al.[13] and Agha et al.[14] [Table 9].
    Table 9: Comparison of specificity of tests with other studies

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      Conclusion Top


    Fungal infection of nails is a problem which is difficult to reliably diagnose clinically due to presence of other diagnostic considerations. Moreover, the treatment of OM is prolonged and has potential side effects. Hence, a definitive diagnosis is a must.

    KOH scraping and FC were two methods used till recently. Both of which have relatively low sensitivity and specificity.

    In our study, we have shown that using PAS on nail scraping can yield higher diagnostic sensitivity with no loss of specificity compared to KOH scrapings.

    We recommend that PAS could replace KOH as a good screening test for the diagnosis of OM. The diagnostic accuracy can be improved by a combination of KOH with HP/PAS.

    Financial support and sponsorship

    Nil.

    Conflicts of interest

    There are no conflicts of interest.



     
      References Top

    1.
    Elewski BE. Onychomycosis: Pathogenesis, diagnosis, and management. Clin Microbiol Rev 1998;11:415-29.  Back to cited text no. 1
        
    2.
    Jesudanam TM, Rao GR, Lakshmi DJ, Kumari GR. Onychomycosis: A significant medical problem. Indian J Dermatol Venereol Leprol 2002;68:326-9.  Back to cited text no. 2
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    3.
    Sen A, Bhunia D, Datta PK, Ray A, Banerjee P. A study of onychomycosis at a tertiary care hospital in eastern Bihar. Indian J Dermatol 2018;63:141-6.  Back to cited text no. 3
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    Garg A, Venkatesh V, Singh M, Pathak KP, Kaushal GP, Agrawal SK. Onychomycosis in central India: A clinicoetiologic correlation. Int J Dermatol 2004;43:498-502.  Back to cited text no. 4
        
    5.
    Grover S. Clinico-mycological evaluation of onychomycosis at Bangalore and Jorhat. Indian J Dermatol Venereol Leprol 2003;69:284-6.  Back to cited text no. 5
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    6.
    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.  Back to cited text no. 6
        
    7.
    Leelavathi M, Tzar MN. Nail sampling technique and its interpretation. Malays Fam Physician 2011;6:58-9.  Back to cited text no. 7
        
    8.
    Bokhari MA, Hussain I, Jahangir M, Haroon TS, Aman S, Khurshid K. Onychomycosis in Lahore, Pakistan. Int J Dermatol 1999;38:591-5.  Back to cited text no. 8
        
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    Rigopoulos D, Katsiboulas V, Koumantaki E, Emmanouil P, Papanicolaou A, Katsambas A. Epidemiology of onychomycosis in southern Greece. Int J Dermatol 1998;37:925-8.  Back to cited text no. 9
        
    10.
    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.  Back to cited text no. 10
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    11.
    Wilsmann-Theis D, Sareika F, Bieber T, Schmid-Wendtner M, Wenzel J. New reasons for histopathological nail-clipping examination in the diagnosis of onychomycosis. J Eur Acad Dermatol Venereol 2011;25:235-7.  Back to cited text no. 11
        
    12.
    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.  Back to cited text no. 12
        
    13.
    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.  Back to cited text no. 13
        
    14.
    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.  Back to cited text no. 14
        
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    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.  Back to cited text no. 15
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    Begari V, Pathakumari P, Takalkar AA. Comparative evaluation of KOH mount, fungal culture and PAS staining in onychomycosis. Int J Res Dermatol 2019;5:554-58.  Back to cited text no. 16
        
    17.
    Alkhayat H, Al-Sulaili N, O’Brein E, McCuaig C, Watters K. The PAS stain for routine diagnosis of onychomycosis. Bahrain Med Bull 2009;31:1-7.  Back to cited text no. 17
        
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    Machler BC, Kirsner RS, Elgart GW. Routine histologic examination for the diagnosis of onychomycosis: An evaluation of sensitivity and specificity. Cutis1998;61:217-9.  Back to cited text no. 18
        


        Figures

      [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6], [Figure 7], [Figure 8]
     
     
        Tables

      [Table 1], [Table 2], [Table 3], [Table 4], [Table 5], [Table 6], [Table 7], [Table 8], [Table 9]



     

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